Transglutaminase variants

ABSTRACT

The present invention provides engineered transglutaminase enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, methods of producing these enzymes, and methods of using the engineered transglutaminase enzymes.

The present application is a divisional of and claims priority toco-pending U.S. patent application Ser. No. 16/652,941, filed Apr. 1,2020, which is a national stage application filed under 35 USC § 371 andclaims priority to international application to PCT InternationalApplication No. PCT/US18/59049, filed Nov. 2, 2018, which claimspriority to U.S. Prov. Pat. Appln. Ser. No. 62/582,593, filed Nov. 7,2017, all of which are incorporated by reference, herein, in theirentireties, for all purposes.

FIELD OF THE INVENTION

The present invention provides engineered transglutaminase enzymes,polynucleotides encoding the enzymes, compositions comprising theenzymes, methods of producing these enzymes, and methods of using theengineered transglutaminase enzymes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-164WO1UD1_ST25.txt”, a creation date of Mar. 28, 2022,and a size of 1,896 kilobytes. The Sequence Listing filed via EFS-Web ispart of the specification and is incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Transglutaminases (TGase; EP2.3.2.13)(R-gluaminyl-peptide-aminase-gamma-glutamyltransferase)comprise an enzyme family that catalyze post-translational modificationsin proteins, producing covalent amide bonds between a primary aminegroup in a polyamine or lysine (i.e., an amine donor) and agamma-carboxyamide group of the glutamyl residue of some proteins andpolypeptides (i.e., an amine acceptor). The result of this enzymaticaction include modification of the protein's conformation and/orextensive conformation changes resulting from the bonding of the sameand different proteins to produce high molecular weight conjugates.These enzymes find use various applications, including in the food,cosmetic, textile, and pharmaceutical industries.

SUMMARY OF THE INVENTION

The present invention provides engineered transglutaminase enzymes,polynucleotides encoding the enzymes, compositions comprising theenzymes, and methods of using the engineered transglutaminase enzymes.

The present invention provides engineered transglutaminases having atleast 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NO: 2, 6, 34, and/or 256. In someembodiments, the engineered transglutaminases have at least 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toSEQ ID NO:6, and at least one substitution or substitution set at one ormore positions selected from positions 79, 101, 101/201/212/287,101/201/285, 101/287, and 327, wherein said positions are numbered withreference to SEQ ID NO:6. In some embodiments, the engineeredtransglutaminases have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to SEQ ID NO:2, and at least onesubstitution or substitution set at one or more positions selected frompositions 48, 48/67/70, 48/67/70/181/203/256, 48/67/70/181/256/345,48/67/70/181/296/345/373, 48/67/70/203/256/296/345,48/67/70/203/256/345/354/373, 48/67/70/203/345, 48/67/70/256,48/67/70/256/296/345/373, 48/67/203/256/296/373, 48/67/203/256/345,48/70/170/203, 48/70/203/254/296/343, 48/70/203/256/345/373,48/70/203/256/345, 48/70/203/373, 48/170/203, 48/170/203/254/296/346,48/170/203/254/296/346/373, 48/170/203/254/346/373, 48/170/203/254/346,48/170/203/296/343/346, 48/170/203/296/346/373, 48/170/203/343/346,48/170/203/346, 48/170/203/346/373, 48/170/203/373, 48/170/254,48/170/296, 48/170/296/343/346, 48/170/343/346, 48/181,48/181/203/256/345, 48/181/203/345, 48/181/256/296/345, 48/181/296,48/181/296/345, 48/203, 48/203/254/296, 48/203/254/296/343/373,48/203/254/296/346/373, 48/203/254/346, 48/203/254/346/373, 48/203/256,48/203/256/296/345, 48/203/296/343/346/373, 48/203/296/343/373,48/203/296/346, 48/203/296/346/373, 48/203/343/346, 48/203/343/346/373,48/203/345, 48/203/346, 48/203/346/373, 48/254/296, 48/254/346, 48/256,48/256/296, 48/256/296/345, 48/296/345, 48/296/373, 48/343/346,48/345/373, 67/256, 67/296/345, 68/74/190/215/346,68/136/215/255/282/297/346, 68/136/215/297/346, 68/136/234,68/158/174/234/282/297/346, 68/158/215/297/346, 68/215/297/346, 68/234,68/282/297/346, 68/297/346, 74/136/174/282/346, 74/136/174/297/346,74/136/346, 74/158/255/297, 74/255/346, 74/346,136/158/190/215/255/297/346, 136/158/215/297/346,136/174/215/255/282/297/346, 136/190/215/297/346, 136/215/234/282/297,136/215/234/297/346, 136/215/297, 136/297/346, 158/215/255/346,158/215/346, 170/203/254/296/343/346, 170/203/254/343/373,170/203/343/346, 174/190/234/297/346, 174/215/234/297/346,174/215/255/297/346, 174/282/297/346, 190/255/282/346, 190/297/346,203/296, 203/343, 203/343/346, 203/346, 215/255/297/346,215/234/297/346, 215/255/297/346, 215/297, 215/297/346, 215/346,234/255/346, 255/297/346, 255/346, 297/346, 343/346/373, and 346,wherein said positions are numbered with reference to SEQ ID NO:2. Insome additional embodiments, the engineered transglutaminases have atleast 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NO: 2, and at least one substitution orsubstitution set at one or more positions selected from33/67/70/181/203/256/296/373, 36/48/203/254/346,48/67/70/181/203/256/296/373, 48/67/70/203/256/296/373,48/67/181/203/256/296/373, 48/67/181/203/256/373, 48/67/181/256/296,48/67/203/256/296/373/378, 48/67/203/256/373, 48/67/203/296/373,48/67/256/296/373, 48/70/181/203/256/296/373, 48/70/181/203/256/373,48/70/181/203/296/373, 48/70/203/256/296/373, 48/70/203/256/373,48/70/203/296, 48/70/203/296/373, 48/70/203/373, 48/70/256/296/373,48/70/296/373, 48/176/203/254/346/373, 48/181/203/256/296/373,48/181/203/256/373, 48/181/203/296, 48/181/203/373, 48/181/256/296/373,48/203/254, 48/203/254/343, 48/203/254/343/346/373,48/203/254/343/355/373, 48/203/254/343/373, 48/203/254/346/373,48/203/254/373, 48/203/256/296, 48/203/256/296/373, 48/203/256/373,48/203/296/373, 48/203/296/373/374, 48/203/343/373, 48/203/373, 48/254,48/254/343/346/373, 48/254/343/373, 48/254/346/373, 48/254/373,48/256/296/373, 48/256/373, 48/373, 67/70/181/203/256/296/373,67/70/181/256/296/373, 67/70/181/373, 67/181/203/256/296,67/181/203/256/296/373, 67/181/203/256/373, 67/203/256/296/373,67/256/296/373, 70/181/203/256/296/373, 70/181/203/296/373, 70/203,70/203/256/296/373, 70/203/256/373, 70/203/296/373,74/136/215/234/282/297/346, 74/136/215/234/282/346, 74/136/215/234/297,74/136/215/234/297/343/346, 74/136/215/234/297/346, 74/136/215/234/346,74/136/215/282/297/346, 74/136/215/282/346, 74/136/215/297/346,74/136/215/346, 74/136/234/282/297/346, 74/136/234/346,74/136/282/297/346, 74/215, 74/215/234/282/297/346, 74/215/282/297/346,74/215/346, 136/215/234/282/297/346, 136/215/282/297,136/215/282/297/346, 136/215/282/346, 136/215/297/346, 136/215/346,136/234/297, 136/234/297/346, 136/234/346, 136/282/297, 181/203/256,181/203/256/296, 181/203/256/296/373, 181/203/256/373, 181/203/296/373,181/203/373, 181/256/296/373, 181/296, 203/224/254/373, 203/254,203/254/343/346/373, 203/254/343/373, 203/254/346, 203/254/346/373,203/254/373, 203/346/373, 203/373, 203/209/256/373, 203/256,203/256/296, 203/256/296/320/373, 203/256/296/373, 203/256/296/373/386,203/256/373, 203/296/373, 203/373, 215/234/282/297/346, 215/234/282/346,215/234/346, 234/282/346, 254, 254/346, 254/346/373, 254/373, 256/296,256/296/373, 256/373, 282/297/346, 343/373, and 373, wherein saidpositions are numbered with reference to SEQ ID NO: 2. In some furtherembodiments, the engineered transglutaminases have at least 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toSEQ ID NO: 34, and at least one substitution or substitution set at oneor more positions selected from 48/49, 49, 50, 50, 331, 291, 292, 330,and 331, wherein said positions are numbered with reference to SEQ IDNO: 34. In yet some additional embodiments, the engineeredtransglutaminases have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to SEQ ID NO: 256, and at leastone substitution or substitution set at one or more positions selectedfrom 27/48/67/70/74/234/256/282/346/373,27/48/67/70/136/203/215/256/282/346/373, 27/48/67/70/346/373,27/48/67/74/203/256/346/373, 27/67/234/296/373, 45/287/328/333,45/292/328, 48, 48/284/292/333, 48/287/292/297, 48/287/297/328/333,48/292, 48/292/297, 48/49/50/292/331, 48/49/50/292, 48/49/50/331,48/49/330/331, 48/49/50/349, 48/49/50/291/292/331, 48/49/50/292/331,48/67/70/203/215/234/256/346, 48/67/70/234/256/282/297/346,48/67/70/346, 48/67/74/203/234/256/282/346/373,48/67/74/234/297/346/373, 48/67/74/346, 48/67/203/346/373,48/67/234/256/297/346/373, 48/67/234/256/346/373,48/67/215/282/297/346/373, 48/67/346/373, 48/70/74/297/346/373,48/70/203/215/256/282/346/373, 48/70/215/234/256/346/373,48/74/203/234/256/346/373, 48/74/234/256/297/346/373,48/136/256/346/373, 48/203/234/256/297/346/373, 48/203/234/256/346/373,48/203/234/346/373, 48/203/296/373, 48/215/234/346/373, 48/215/346/373,48/234/256/296/346/373, 48/234/256/346/373, 48/256/373, 49/50/292/331,49/50/292/331/349, 49/50/331, 49/50/331/349, 50,67/70/74/136/203/215/256/346/373, 67/70/74/203/215/234/346/373,67/70/74/215/234/297/346/373, 67/70/74/215/256/373,67/70/136/203/297/346/373, 67/70/203/215/256/346/373, 67/70/203/373,67/70/215, 67/74/136, 67/74/203/234/256, 67/74/215/256/297/346/373,67/74/215/346/373, 67/74/256/346/373, 67/136/203/215/256/346/373,67/136/203/256/346/373, 67/203/234/256/346/373, 67/203/297/346/373,67/215/234/297/346/373, 67/297/346, 70/74/203/215/346/373, 136,136/346/373, 203/234/346, 203/234/346/373, 203/373, 234/282, 287,234/346/373, 287/292, 287/292/295/297, 287/292/297, 287/295/297,287/330/333, 292, 292/297, 292/330/331, 292/330/331, 292/331,292/331/349, 292/349, 295, 295/297/333, 297/328, 297/373, 328/333, 330,330/331, 331, 331/349, 333, 346/373, and 373, wherein said positions arenumbered with reference to SEQ ID NO: 256. In some embodiments, theengineered transglutaminases comprise a polypeptide sequence comprisinga sequence having at least 90% sequence identity to SEQ ID NO:2, 6, 34,and/or 256. In some alternative embodiments, the engineeredtransglutaminases comprise a polypeptide sequence comprising a sequencehaving at least 95% sequence identity to SEQ ID NO:2, 6, 34, and/or 256.In yet some additional embodiments, the engineered transglutaminasescomprise a polypeptide sequence set forth in SEQ ID NO:2, 6, 34, or 256.In yet some additional embodiments, the engineered transglutaminasescomprise a polypeptide sequence encoding a variant provided in Table8.1, 9.1, 9.2, 10.1, and/or 11.1. In some further embodiments, theengineered transglutaminase comprises a polypeptide sequence selectedfrom the even-numbered sequences set from in SEQ ID NOS: 4 to 756.

The present invention also provides engineered polynucleotide sequencesencoding the engineered transglutaminases provided herein. In someembodiments, the engineered polynucleotide sequences comprisepolynucleotide sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical to a sequence selected fromthe odd-numbered sequences set forth in SEQ ID NOS: 3 to 755. Thepresent invention also provides vectors comprising the engineeredpolynucleotide sequences provided herein. In some embodiments, thevectors further comprise at least one control sequence. The presentinvention also provides host cells comprising the vectors providedherein.

The present invention also provides methods for producing the engineeredtransglutaminases provided herein, comprising culturing a host cellunder conditions that at least one engineered transglutaminase isproduced by said host cell. In some embodiments, the host cell producesan engineered transglutaminase. In some embodiments, the methods furthercomprise the step of recovering said engineered transglutaminaseproduced by said host cell.

The present invention also provides engineered transglutaminases capableof modifying a free amine in insulin in the presence of a glutaminedonor. In some embodiments, the engineered transglutaminases providedherein are capable of modifying a glutamine in insulin in the presenceof a lysine donor.

The present invention also provides methods of modifying insulincomprising: providing insulin and at least one engineeredtransglutaminase provided herein, combining said insulin, glutamine, andat least one engineered transglutaminase under conditions such that saidinsulin is modified. The present invention also provides methods ofmodifying insulin comprising: providing insulin and at least oneengineered transglutaminase provided herein, combining said insulin,lysine, and at least one engineered transglutaminase under conditionssuch that said insulin is modified.

DESCRIPTION OF THE INVENTION

The present invention provides engineered transglutaminase enzymes,polynucleotides encoding the enzymes, compositions comprising theenzymes, and methods of using the engineered transglutaminase enzymes.

The transglutaminase variants provided herein are engineered from theStreptomyces mobaraensis transglutaminase of SEQ ID NO:2, in whichvarious modifications have been introduced to generate improvedenzymatic properties as described in detail below.

For the descriptions provided herein, the use of the singular includesthe plural (and vice versa) unless specifically stated otherwise. Forinstance, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Both the foregoing general description, including the drawings, and thefollowing detailed description are exemplary and explanatory only andare not restrictive of this disclosure. Moreover, the section headingsused herein are for organizational purposes only and not to be construedas limiting the subject matter described.

Definitions

As used herein, the following terms are intended to have the followingmeanings. In reference to the present disclosure, the technical andscientific terms used in the descriptions herein will have the meaningscommonly understood by one of ordinary skill in the art, unlessspecifically defined otherwise. Accordingly, the following terms areintended to have the following meanings. In addition, all patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,fermentation, microbiology, and related fields, which are known to thoseof skill in the art. Unless defined otherwise herein, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods and materials are described.Indeed, it is intended that the present invention not be limited to theparticular methodology, protocols, and reagents described herein, asthese may vary, depending upon the context in which they are used. Theheadings provided herein are not limitations of the various aspects orembodiments of the present invention that can be had by reference to thespecification as a whole. Accordingly, the terms defined below are morefully defined by reference to the specification as a whole.

Nonetheless, in order to facilitate understanding of the presentinvention, a number of terms are defined below. Numeric ranges areinclusive of the numbers defining the range. Thus, every numerical rangedisclosed herein is intended to encompass every narrower numerical rangethat falls within such broader numerical range, as if such narrowernumerical ranges were all expressly written herein. It is also intendedthat every maximum (or minimum) numerical limitation disclosed hereinincludes every lower (or higher) numerical limitation, as if such lower(or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and“the” include the plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “host cell” includes aplurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation and amino acid sequences are written left to rightin amino to carboxy orientation, respectively.

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably herein to denote a polymer of at least two aminoacids covalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids.

As used herein, “polynucleotide” and “nucleic acid” refer to two or morenucleosides that are covalently linked together. The polynucleotide maybe wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of”2′ deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), itmay include one or more modified and/or synthetic nucleobases (e.g.,inosine, xanthine, hypoxanthine, etc.). Preferably, such modified orsynthetic nucleobases will be encoding nucleobases.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are known to those of skill in the art.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. In some embodiments, thepolynucleotides encoding the transglutaminase enzymes may be codonoptimized for optimal production from the host organism selected forexpression. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding thetransglutaminase enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

As used herein, “preferred, optimal, high codon usage bias codons”refers interchangeably to codons that are used at higher frequency inthe protein coding regions than other codons that code for the sameamino acid. The preferred codons may be determined in relation to codonusage in a single gene, a set of genes of common function or origin,highly expressed genes, the codon frequency in the aggregate proteincoding regions of the whole organism, codon frequency in the aggregateprotein coding regions of related organisms, or combinations thereof.Codons whose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (See e.g., GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney,Bioinform., 14:372-73 [1998]; Stenico et al., Nucleic Acids Res.,222:437-46 [1994]; and Wright, Gene 87:23-29 [1990]). Codon usage tablesare available for a growing list of organisms (See e.g., Wada et al.,Nucleic Acids Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. AcidsRes., 28:292 [2000]; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” Neidhardt, et al. (eds.), ASM Press,Washington D.C., [1996], p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (See e.g., Uberbacher, Meth.Enzymol., 266:259-281 [1996]; Tiwari et al., Comput. Appl. Biosci.,13:263-270 [1997]).

As used herein, “control sequence” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide and/or polypeptide of the present invention. Each controlsequence may be native or foreign to the polynucleotide of interest.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator.

As used herein, “operably linked” is defined herein as a configurationin which a control sequence is appropriately placed (i.e., in afunctional relationship) at a position relative to a polynucleotide ofinterest such that the control sequence directs or regulates theexpression of the polynucleotide and/or polypeptide of interest.

As used herein, “promoter sequence” refers to a nucleic acid sequencethat is recognized by a host cell for expression of a polynucleotide ofinterest, such as a coding sequence. The control sequence may comprisean appropriate promoter sequence. The promoter sequence containstranscriptional control sequences, which mediate the expression of apolynucleotide of interest. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

As used herein, “naturally occurring” and “wild-type” refers to the formfound in nature. For example, a naturally occurring or wild-typepolypeptide or polynucleotide sequence is a sequence present in anorganism that can be isolated from a source in nature and which has notbeen intentionally modified by human manipulation.

As used herein, “non-naturally occurring,” “engineered,” and“recombinant” when used in the present disclosure with reference to(e.g., a cell, nucleic acid, or polypeptide), refers to a material, or amaterial corresponding to the natural or native form of the material,that has been modified in a manner that would not otherwise exist innature. In some embodiments the material is identical to naturallyoccurring material, but is produced or derived from synthetic materialsand/or by manipulation using recombinant techniques. Non-limitingexamples include, among others, recombinant cells expressing genes thatare not found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise expressed at a different level.

As used herein, “percentage of sequence identity,” “percent identity,”and “percent identical” refer to comparisons between polynucleotidesequences or polypeptide sequences, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whicheither the identical nucleic acid base or amino acid residue occurs inboth sequences or a nucleic acid base or amino acid residue is alignedwith a gap to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Determination of optimal alignment and percentsequence identity is performed using the BLAST and BLAST 2.0 algorithms(See e.g., Altschul et al., J. Mol. Biol. 215: 403-410 [1990]; andAltschul et al., Nucl. Acids Res. 3389-3402 [1977]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length Win the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as, the neighborhood word score threshold(Altschul et al., supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915 [1989]).

Numerous other algorithms are available and known in the art thatfunction similarly to BLAST in providing percent identity for twosequences. Optimal alignment of sequences for comparison can beconducted using any suitable method known in the art (e.g., by the localhomology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 [1981];by the homology alignment algorithm of Needleman and Wunsch, J. Mol.Biol. 48:443 [1970]; by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; and/or bycomputerized implementations of these algorithms [GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package]), or by visualinspection, using methods commonly known in the art. Additionally,determination of sequence alignment and percent sequence identity canemploy the BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using the default parameters provided.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity and 89 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of at least 20 residue positions,frequently over a window of at least 30-50 residues, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to a sequence that includes deletions or additions which total20 percent or less of the reference sequence over the window ofcomparison. In specific embodiments applied to polypeptides, the term“substantial identity” means that two polypeptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80 percent sequence identity, preferably atleast 89 percent sequence identity, at least 95 percent sequenceidentity or more (e.g., 99 percent sequence identity). In some preferredembodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As used herein, “reference sequence” refers to a defined sequence towhich another sequence is compared. A reference sequence may be a subsetof a larger sequence, for example, a segment of a full-length gene orpolypeptide sequence. Generally, a reference sequence is at least 20nucleotide or amino acid residues in length, at least 25 residues inlength, at least 50 residues in length, or the full length of thenucleic acid or polypeptide. Since two polynucleotides or polypeptidesmay each (1) comprise a sequence (i.e., a portion of the completesequence) that is similar between the two sequences, and (2) may furthercomprise a sequence that is divergent between the two sequences,sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a comparison window to identify and compare localregions of sequence similarity. The term “reference sequence” is notintended to be limited to wild-type sequences, and can includeengineered or altered sequences. For example, in some embodiments, a“reference sequence” can be a previously engineered or altered aminoacid sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acids residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to,” “reference to,” and “relative to”when used in the context of the numbering of a given amino acid orpolynucleotide sequence refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered transglutaminase, can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or polynucleotide sequence is made withrespect to the reference sequence to which it has been aligned. As usedherein, a reference to a residue position, such as “Xn” as furtherdescribed below, is to be construed as referring to “a residuecorresponding to”, unless specifically denoted otherwise. Thus, forexample, “X94” refers to any amino acid at position 94 in a polypeptidesequence.

As used herein, “improved enzyme property” refers to a transglutaminasethat exhibits an improvement in any enzyme property as compared to areference transglutaminase. For the engineered transglutaminasepolypeptides described herein, the comparison is generally made to thewild-type transglutaminase enzyme, although in some embodiments, thereference transglutaminase is another improved engineeredtransglutaminase. Enzyme properties for which improvement is desirableinclude, but are not limited to, enzymatic activity (which can beexpressed in terms of percent conversion of the substrate at a specifiedreaction time using a specified amount of transglutaminase),chemoselectivity, thermal stability, solvent stability, pH activityprofile, cofactor requirements, refractoriness to inhibitors (e.g.,product inhibition), stereospecificity, and stereoselectivity (includingenantioselectivity).

As used herein, “increased enzymatic activity” refers to an improvedproperty of the engineered transglutaminase polypeptides, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of transglutaminase) as compared to the referencetransglutaminase enzyme. Exemplary methods to determine enzyme activityare provided in the Examples. Any property relating to enzyme activitymay be affected, including the classical enzyme properties of K_(m),V_(max) or k_(cat), changes of which can lead to increased enzymaticactivity. Improvements in enzyme activity can be from about 1.5 timesthe enzymatic activity of the corresponding wild-type transglutaminaseenzyme, to as much as 2 times. 5 times, 10 times, 20 times, 25 times, 50times, 75 times, 100 times, or more enzymatic activity than thenaturally occurring transglutaminase or another engineeredtransglutaminase from which the transglutaminase polypeptides werederived. In specific embodiments, the engineered transglutaminase enzymeexhibits improved enzymatic activity in the range of 1.5 to 50 times,1.5 to 100 times greater than that of the parent transglutaminaseenzyme. It is understood by the skilled artisan that the activity of anyenzyme is diffusion limited such that the catalytic turnover rate cannotexceed the diffusion rate of the substrate, including any requiredcofactors. The theoretical maximum of the diffusion limit, ork_(cat)/K_(m), is generally about 10⁸ to 10⁹ (M⁻¹ s⁻¹). Hence, anyimprovements in the enzyme activity of the transglutaminase will have anupper limit related to the diffusion rate of the substrates acted on bythe transglutaminase enzyme. Transglutaminase activity can be measuredby any one of standard assays available in the art (e.g., hydroxymateassays). Comparisons of enzyme activities are made using a definedpreparation of enzyme, a defined assay under a set condition, and one ormore defined substrates, as further described in detail herein.Generally, when lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

As used herein, “increased enzymatic activity” and “increased activity”refer to an improved property of an engineered enzyme, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of transglutaminase) as compared to a reference enzymeas described herein. Any property relating to enzyme activity may beaffected, including the classical enzyme properties of K_(m), V_(max) ork_(cat), changes of which can lead to increased enzymatic activity.Comparisons of enzyme activities are made using a defined preparation ofenzyme, a defined assay under a set condition, and one or more definedsubstrates, as further described in detail herein. Generally, whenenzymes in cell lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

As used herein, “conversion” refers to the enzymatic transformation of asubstrate to the corresponding product.

As used herein “percent conversion” refers to the percent of thesubstrate that is converted to the product within a period of time underspecified conditions. Thus, for example, the “enzymatic activity” or“activity” of a transglutaminase polypeptide can be expressed as“percent conversion” of the substrate to the product.

As used herein, “chemoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one product over another.

As used herein, “thermostable” and “thermal stable” are usedinterchangeably to refer to a polypeptide that is resistant toinactivation when exposed to a set of temperature conditions (e.g.,40-80° C.) for a period of time (e.g., 0.5-24 hrs) compared to theuntreated enzyme, thus retaining a certain level of residual activity(e.g., more than 60% to 80%) after exposure to elevated temperatures.

As used herein, “solvent stable” refers to the ability of a polypeptideto maintain similar activity (e.g., more than e.g., 60% to 80%) afterexposure to varying concentrations (e.g., 5-99%) of solvent (e.g.,isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone,toluene, butylacetate, methyl tert-butylether, etc.) for a period oftime (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “pH stable” refers to a transglutaminase polypeptidethat maintains similar activity (e.g., more than 60% to 80%) afterexposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time(e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “thermo- and solvent stable” refers to atransglutaminase polypeptide that is both thermostable and solventstable.

As used herein, “hydrophilic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity of lessthan zero according to the normalized consensus hydrophobicity scale ofEisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]).Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser(S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K)and L-Arg (R).

As used herein, “acidic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pK value of lessthan about 6 when the amino acid is included in a peptide orpolypeptide. Acidic amino acids typically have negatively charged sidechains at physiological pH due to loss of a hydrogen ion. Geneticallyencoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, “basic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pK value ofgreater than about 6 when the amino acid is included in a peptide orpolypeptide. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, “polar amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Genetically encoded polar amino acids include L-Asn (N),L-Gln (Q), L-Ser (S) and L-Thr (T).

As used herein, “hydrophobic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity ofgreater than zero according to the normalized consensus hydrophobicityscale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142[1984]). Genetically encoded hydrophobic amino acids include L-Pro (P),L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala(A) and L-Tyr (Y).

As used herein, “aromatic amino acid or residue” refers to a hydrophilicor hydrophobic amino acid or residue having a side chain that includesat least one aromatic or heteroaromatic ring. Genetically encodedaromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W).Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H)it is sometimes classified as a basic residue, or as an aromatic residueas its side chain includes a heteroaromatic ring, herein histidine isclassified as a hydrophilic residue or as a “constrained residue” (seebelow).

As used herein, “constrained amino acid or residue” refers to an aminoacid or residue that has a constrained geometry. Herein, constrainedresidues include L-Pro (P) and L-His (H). Histidine has a constrainedgeometry because it has a relatively small imidazole ring. Proline has aconstrained geometry because it also has a five membered ring.

As used herein, “non-polar amino acid or residue” refers to ahydrophobic amino acid or residue having a side chain that is unchargedat physiological pH and which has bonds in which the pair of electronsshared in common by two atoms is generally held equally by each of thetwo atoms (i.e., the side chain is not polar). Genetically encodednon-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile(I), L-Met (M) and L-Ala (A).

As used herein, “aliphatic amino acid or residue” refers to ahydrophobic amino acid or residue having an aliphatic hydrocarbon sidechain. Genetically encoded aliphatic amino acids include L-Ala (A),L-Val (V), L-Leu (L) and L-Ile (I). It is noted that cysteine (or“L-Cys” or “[C]”) is unusual in that it can form disulfide bridges withother L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containingamino acids. The “cysteine-like residues” include cysteine and otheramino acids that contain sulfhydryl moieties that are available forformation of disulfide bridges. The ability of L-Cys (C) (and otheramino acids with —SH containing side chains) to exist in a peptide ineither the reduced free —SH or oxidized disulfide-bridged form affectswhether L-Cys (C) contributes net hydrophobic or hydrophilic characterto a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29according to the normalized consensus scale of Eisenberg (Eisenberg etal., 1984, supra), it is to be understood that for purposes of thepresent disclosure, L-Cys (C) is categorized into its own unique group.

As used herein, “small amino acid or residue” refers to an amino acid orresidue having a side chain that is composed of a total three or fewercarbon and/or heteroatoms (excluding the α-carbon and hydrogens). Thesmall amino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T)and L-Asp (D).

As used herein, “hydroxyl-containing amino acid or residue” refers to anamino acid containing a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

As used herein, “amino acid difference” and “residue difference” referto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. The positions of amino acid differencesgenerally are referred to herein as “Xn,” where n refers to thecorresponding position in the reference sequence upon which the residuedifference is based. For example, a “residue difference at position X40as compared to SEQ ID NO:2” refers to a difference of the amino acidresidue at the polypeptide position corresponding to position 40 of SEQID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has ahistidine at position 40, then a “residue difference at position X40 ascompared to SEQ ID NO:2” refers to an amino acid substitution of anyresidue other than histidine at the position of the polypeptidecorresponding to position 40 of SEQ ID NO:2. In most instances herein,the specific amino acid residue difference at a position is indicated as“XnY” where “Xn” specified the corresponding position as describedabove, and “Y” is the single letter identifier of the amino acid foundin the engineered polypeptide (i.e., the different residue than in thereference polypeptide). In some instances, the present disclosure alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present disclosure can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X192A/G). The presentdisclosure includes engineered polypeptide sequences comprising one ormore amino acid differences that include either/or both conservative andnon-conservative amino acid substitutions. The amino acid sequences ofthe specific recombinant carbonic anhydrase polypeptides included in theSequence Listing of the present disclosure include an initiatingmethionine (M) residue (i.e., M represents residue position 1). Theskilled artisan, however, understands that this initiating methionineresidue can be removed by biological processing machinery, such as in ahost cell or in vitro translation system, to generate a mature proteinlacking the initiating methionine residue, but otherwise retaining theenzyme's properties. Consequently, the term “amino acid residuedifference relative to SEQ ID NO:2 at position Xn” as used herein mayrefer to position “Xn” or to the corresponding position (e.g., position(X−1)n) in a reference sequence that has been processed so as to lackthe starting methionine.

As used herein, the phrase “conservative amino acid substitutions”refers to the interchangeability of residues having similar side chains,and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with a hydroxyl side chain is substituted with anotheramino acid with a hydroxyl side chain (e.g., serine and threonine); anamino acid having aromatic side chains is substituted with another aminoacid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basic side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. Exemplary conservative substitutions are provided inTable 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions ResiduePotential Conservative Substitutions A, L, V, I Other aliphatic (A, L,V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A, L, V,I, G, M) D, E Other acidic (D, E) K, R Other basic (K, R) N, Q, S, TOther polar H, Y, W, F Other aromatic (H, Y, W, F) C, P Non-polar

As used herein, the phrase “non-conservative substitution” refers tosubstitution of an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification of the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thepolypeptide while retaining enzymatic activity and/or retaining theimproved properties of an engineered enzyme. Deletions can be directedto the internal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

As used herein, “insertion” refers to modification of the polypeptide byaddition of one or more amino acids to the reference polypeptide. Insome embodiments, the improved engineered transglutaminase enzymescomprise insertions of one or more amino acids to the naturallyoccurring transglutaminase polypeptide as well as insertions of one ormore amino acids to engineered transglutaminase polypeptides. Insertionscan be in the internal portions of the polypeptide, or to the carboxy oramino terminus. Insertions as used herein include fusion proteins as isknown in the art. The insertion can be a contiguous segment of aminoacids or separated by one or more of the amino acids in the naturallyoccurring polypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Insome embodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant transglutaminaseslisted in the Tables provided in the Examples.

As used herein, “fragment” refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can typically have about 80%, about 90%, about 95%,about 98%, or about 99% of the full-length transglutaminase polypeptide,for example the polypeptide of SEQ ID NO:2. In some embodiments, thefragment is “biologically active” (i.e., it exhibits the same enzymaticactivity as the full-length sequence).

As used herein, “isolated polypeptide” refers to a polypeptide that issubstantially separated from other contaminants that naturally accompanyit (e.g., proteins, lipids, and polynucleotides). The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., host cell orin vitro synthesis). The improved transglutaminase enzymes may bepresent within a cell, present in the cellular medium, or prepared invarious forms, such as lysates or isolated preparations. As such, insome embodiments, the engineered transglutaminase polypeptides of thepresent disclosure can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” refers to a compositionin which the polypeptide species is the predominant species present(i.e., on a molar or weight basis it is more abundant than any otherindividual macromolecular species in the composition), and is generallya substantially purified composition when the object species comprisesat least about 50 percent of the macromolecular species present by moleor % weight. Generally, a substantially pure engineered transglutaminasepolypeptide composition comprises about 60% or more, about 70% or more,about 80% or more, about 90% or more, about 91% or more, about 92% ormore, about 93% or more, about 94% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more, or about 99% of allmacromolecular species by mole or % weight present in the composition.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated improved transglutaminase polypeptide is a substantiallypure polypeptide composition.

As used herein, when used in reference to a nucleic acid or polypeptide,the term “heterologous” refers to a sequence that is not normallyexpressed and secreted by an organism (e.g., a wild-type organism). Insome embodiments, the term encompasses a sequence that comprises two ormore subsequences which are not found in the same relationship to eachother as normally found in nature, or is recombinantly engineered sothat its level of expression, or physical relationship to other nucleicacids or other molecules in a cell, or structure, is not normally foundin nature. For instance, a heterologous nucleic acid is typicallyrecombinantly produced, having two or more sequences from unrelatedgenes arranged in a manner not found in nature (e.g., a nucleic acidopen reading frame (ORF) of the invention operatively linked to apromoter sequence inserted into an expression cassette, such as avector). In some embodiments, “heterologous polynucleotide” refers toany polynucleotide that is introduced into a host cell by laboratorytechniques, and includes polynucleotides that are removed from a hostcell, subjected to laboratory manipulation, and then reintroduced into ahost cell.

As used herein, “suitable reaction conditions” refer to those conditionsin the biocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which a transglutaminase polypeptide of thepresent disclosure is capable of modifying a substrate of interest. Insome embodiments, the transglutaminases cross-link substitutedglutamines and substituted lysines in various substrates, including lowmolecular weight substrates and proteins. In some embodiments, thetransglutaminases of the present invention are capable of site specificmodification of biological macromolecules. Exemplary “suitable reactionconditions” are provided in the present disclosure and illustrated bythe Examples.

As used herein, “loading,” such as in “compound loading,” “enzymeloading,” or “cofactor loading” refers to the concentration or amount ofa component in a reaction mixture at the start of the reaction.

As used herein, “substrate” in the context of a biocatalyst mediatedprocess refers to the compound or molecule acted on by the biocatalyst.

As used herein “product” in the context of a biocatalyst mediatedprocess refers to the compound or molecule resulting from the action ofthe biocatalyst.

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

As used herein, “transglutaminase,” “TG,” “TGase,” and “polypeptide withtransglutaminase activity,” refer to an enzyme having the ability tocatalyze the acyl transfer reaction between the gamma-carboxyamide groupin a peptide/protein (e.g., glutamine residues) and various primaryamines, which act as amine donors. In some embodiments, there is asubstitution reaction of glutamine with glutamic acid by the deamidationof glutamic acid. In some embodiments, lysine is used as the acylacceptor, which results in the enrichment of the protein molecule usedin the reaction. The transfer of acyl onto a lysine residue in apolypeptide chain induces the cross-linking process (i.e., the formationof intra- or inter-molecular cross-links (See e.g., Kieliszek andMisiewicz, supra; and Kashiwagi et al., J. Biol. Chem., 277:44252-44260[2002]). In some embodiments, transglutaminases find use in catalyzingdeamination reactions in the absence of free amine groups, but thepresence of water, which acts as an acyl acceptor. This results insignificant changes in the physical and chemical properties of affectedproteins, including modifications in viscosity, thermostability,elasticity, and resilience (See e.g., Kieliszek and Misiewicz, supra;Motoki and Seguroa, Trends Food Sci. Technol., 9:204-210 [1998]; andKuraishi et al., Food Rev. Intl., 17:221-246 [2001]). Transglutaminasesare known to be widely distributed in various organisms, includinghumans, bacteria, nematodes, yeasts, algae, plants, and lowervertebrates (See e.g., Santos and Tome, Recent Pat. Biotechnol.,3:166-174 [2009]).

As used herein, “transglutamination,” “transamination,” and“transglutaminase reaction” refer to reactions in which thegamma-glutaminyl of glutamine residue from a protein/polypeptide/peptideis transferred to a primary amine or the episilon-amino group of lysineor water, wherein an ammonia molecule is released.

As used herein, “derived from” when used in the context of engineeredtransglutaminase enzymes, identifies the originating transglutaminaseenzyme, and/or the gene encoding such transglutaminase enzyme, uponwhich the engineering was based. For example, the engineeredtransglutaminase enzyme of SEQ ID NO: 296 was obtained by artificiallyevolving, over multiple generations the gene (SEQ ID NO:1) encoding theS. mobaraensis transglutaminase of SEQ ID NO:2.

Thus, this engineered transglutaminase enzyme is “derived from” thenaturally occurring or wild-type transglutaminase of SEQ ID NO: 2.

Transglutaminases

The present invention provides variant transglutaminases developed froma wild-type S. mobaraensis transglutaminase enzyme. S. mobaraensis isalso classified as Strep toverticilliium mobaraese. This enzyme has amolecular weight of about 38 kDa and is calcium independent (See e.g.,Appl. Microbiol. Biotech., 64:447-454 [2004]; and US Pat. Appln. Publ.No. 2010/0099610, incorporated herein by reference).

Transglutaminases have found use in altering the properties of variouspeptides. In some embodiments, the enzyme is used to cross-bind peptidesuseful in the food and dairy industries, as well as in uses involvingphysiologically active peptides, biomedicine, biomaterials, antibodies,the textile industry (e.g., wool and leather), methods for peptideconjugation, linkage of agents to tissue, cosmetics, etc. (See e.g., EP950 665, EP 785 276, WO 2005/070468, WO 2006/134148, WO 2008/102007, WO2009/003732, U.S. Pat. No. 6,013,498, US Pat. Appln. Publ. No.2010/0099610; US Pat. Appln. Publ. No. 2010/0249029; and US Pat. Appln.Publ. No. 2010/0087371, each of which is incorporated by referenceherein; and Sato, Adv. Drug Deliv. Rev., 54:487-504 [2002]; Valdivia, J.Biotechnol., 122:326-333 [2006]; Wada, Biotech. Lett., 23:1367-1372[2001]; Kieliszek and Misiewicz, Folia Microbiol., 59:241-250 [2014];Yokoyama et al., Appl. Microbiol. Biotechol., 64:447-454 [2004]; Washizuet al., Biosci. Biotech. Biochem., 58:82-87 [1994]; Kanaji et al., J.Biol. Chem., 268:11565-11572 [1993]; Ando et al., Agric. Biol. Chem.53:2613-2617 [1989]; Martins et al., Appl. Microbiol. Biotechnol.,98:6957-6964 [2014]; Jerger et al., Angew. Chem. Int., 49:9995-9997[2010]; Grunberg et al., PLoS ONE 8:e60350 [2013]; Mindt et al.,Bioconj. Chem., 19:271-278 [2008]; Lhospice et al., Mol. Pharmaceutics12:1863-1871 [2015]; Dennler et al., Bioconj. Chem., 25:569-578 [2014];and Santos and Tome, Rec. Patents Biotechnol., 3:166-174 [2009], fordiscussion of transglutaminases, their sources, and uses). These enzymesare capable of improving the firmness, viscosity, elasticity, andwater-binding capacity of food and other products.

In some embodiments, the transglutaminase variants provided herein finduse in the food industry for production of foods (e.g., jelly, yogurt,cheese, noodles, chewing gum, candy, baked products, soybean protein,gummy candy, snacks, pickles, meat, and chocolate), while in some otherembodiments, the transglutaminase variants find use in other industries(e.g., textiles, pharmaceuticals, diagnostics, etc.).

In some embodiments, the present invention provides engineeredtransglutaminase polypeptides with amino acid sequences that have atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% or more sequence identity to SEQ ID NO: 2, 6, 34,and/or 256.

In some embodiments, the engineered transglutaminase polypeptidescomprise substitutions at one or more positions selected from 79, 101,101/201/212/287, 101/201/285, 101/287, and 327, wherein the positionsare numbered with reference to SEQ ID NO:6. In some embodiments, theengineered transglutaminase polypeptides comprise one or moresubstitutions selected from 79K, 101G, 101G/201K/212K/287G,101G/201K/285Q, 101G/287G, and 327R, wherein the positions are numberedwith reference to SEQ ID NO:6. In some embodiments, the engineeredtransglutaminase polypeptides comprise one or more substitutionsselected from S79K, Y101G, Y101G/Q201K/R212K/S287G, Y101G/Q201K/R285Q,Y101G/S287G, and G327R, wherein the positions are numbered withreference to SEQ ID NO:6.

In some embodiments, the engineered transglutaminase polypeptidescomprise substitutions at one or more positions selected from 48,48/67/70, 48/67/70/181/203/256, 48/67/70/181/256/345,48/67/70/181/296/345/373, 48/67/70/203/256/296/345,48/67/70/203/256/345/354/373, 48/67/70/203/345, 48/67/70/256,48/67/70/256/296/345/373, 48/67/203/256/296/373, 48/67/203/256/345,48/70/170/203, 48/70/203/254/296/343, 48/70/203/256/345/373,48/70/203/256/345, 48/70/203/373, 48/170/203, 48/170/203/254/296/346,48/170/203/254/296/346/373, 48/170/203/254/346/373, 48/170/203/254/346,48/170/203/296/343/346, 48/170/203/296/346/373, 48/170/203/343/346,48/170/203/346, 48/170/203/346/373, 48/170/203/373, 48/170/254,48/170/296, 48/170/296/343/346, 48/170/343/346, 48/181,48/181/203/256/345, 48/181/203/345, 48/181/256/296/345, 48/181/296,48/181/296/345, 48/203, 48/203/254/296, 48/203/254/296/343/373,48/203/254/296/346/373, 48/203/254/346, 48/203/254/346/373, 48/203/256,48/203/256/296/345, 48/203/296/343/346/373, 48/203/296/343/373,48/203/296/346, 48/203/296/346/373, 48/203/343/346, 48/203/343/346/373,48/203/345, 48/203/346, 48/203/346/373, 48/254/296, 48/254/346, 48/256,48/256/296, 48/256/296/345, 48/296/345, 48/296/373, 48/343/346,48/345/373, 67/256, 67/296/345, 68/74/190/215/346,68/136/215/255/282/297/346, 68/136/215/297/346, 68/136/234,68/158/174/234/282/297/346, 68/158/215/297/346, 68/215/297/346, 68/234,68/282/297/346, 68/297/346, 74/136/174/282/346, 74/136/174/297/346,74/136/346, 74/158/255/297, 74/255/346, 74/346,136/158/190/215/255/297/346, 136/158/215/297/346,136/174/215/255/282/297/346, 136/190/215/297/346, 136/215/234/282/297,136/215/234/297/346, 136/215/297, 136/297/346, 158/215/255/346,158/215/346, 170/203/254/296/343/346, 170/203/254/343/373,170/203/343/346, 174/190/234/297/346, 174/215/234/297/346,174/215/255/297/346, 174/282/297/346, 190/255/282/346, 190/297/346,203/296, 203/343, 203/343/346, 203/346, 215/255/297/346,215/234/297/346, 215/255/297/346, 215/297, 215/297/346, 215/346,234/255/346, 255/297/346, 255/346, 297/346, 343/346/373, and 346,wherein the positions are numbered with reference to SEQ ID NO:2. Insome embodiments, the engineered transglutaminase polypeptides compriseone or more substitutions selected from 48K/70L/203L/254Q/296L/343R,48K/170K/203L, 48K/170K/203L/254Q/296L/346H,48K/170K/203L/254Q/296L/346H/373M, 48K/170K/203L/254Q/346H/373M,48K/170K/203L/254Q/346H, 48K/170K/203L/296L/343R/346H,48K/170K/203L/296L/346H/373M, 48K/170K/203L/343R/346H,48K/170K/203L/346H/373M, 48K/170K/203L/346H, 48K/170K/203L/373M,48K/170K/254Q, 48K/170K/296L, 48K/170K/296L/343R/346H,48K/170K/343R/346H, 48K/203L, 48K/203L/254Q/296L,48K/203L/254Q/296L/343R/373M, 48K/203L/254Q/296L/346H/373M,48K/203L/254Q/346H, 48K/203L/254Q/346H/373M,48K/203L/296L/343R/346H/373M, 48K/203L/296L/343R/373M,48K/203L/296L/346H, 48K/203L/296L/346H/373M, 48K/203L/343R/346H,48K/203L/343R/346H/373M, 48K/203L/346H, 48K/203L/346H/373M,48K/254Q/346H, 48K/343R/346H, 48V, 48V/67E/70G,48V/67E/70G/181K/203V/256G, 48V/67E/70G/181K/256G/345E,48V/67E/70G/181K/296R/345E/373V, 48V/67E/70G/203V/256G/296R/345E,48V/67E/70G/203V/345E, 48V/67E/70G/256G/296R/345E/373V,48V/67E/70N/203V/256G/345E/354H/373L, 48V/67E/70N/256G,48V/67E/203V/256G/296R/373V, 48V/67E/203V/256G/345E, 48K/70D/170K/203L,48V/70G/203V/256G/345E/373V, 48V/70N/203V/256G/345E, 48V/70N/203V/373V,48V/181K, 48V/181K/203V/256G/345E, 48V/181K/203V/345E,48V/181K/256G/296R/345E, 48V/181K/296R, 48V/181K/296R/345E, 48V/203V,48V/203V/256G, 48V/203V/256G/296R/345E, 48V/203V/345E, 48K/254Q/296L,48V/256G, 48V/256G/296R, 48V/256G/296R/345E, 48V/296R/345E,48V/296R/373V, 48V/345E/373L, 67E/256G, 67E/296R/345E,68A/74T/190G/215N/346A, 68A/136Y/215N/255R/282K/297W/346A,68A/136Y/215N/297W/346A, 68A/136Y/234Y,68A/158I/174D/234Y/282K/297W/346A, 68A/158I/215N/297W/346A,68A/215N/297W/346A, 68A/234Y, 68A/282K/297W/346A, 68A/297W/346A,74T/136Y/174D/282K/346A, 74T/136Y/174D/297W/346A, 74T/136Y/346A,74T/158I/255R/297W, 74T/255R/346A, 74T/346A,136Y/158I/190G/215N/255R/297W/346A, 136Y/158I/215N/297W/346A,136Y/174D/215N/255R/282K/297W/346A, 136Y/190G/215N/297W/346A,136Y/215N/234Y/282K/297W, 136Y/215N/234Y/297W/346A, 136Y/215N/297W,136Y/297W/346A, 158I/215N/255R/346A, 158I/215N/346A,170K/203L/254Q/296L/343R/346H, 170K/203L/254Q/343R/373M,170K/203L/343R/346H, 174D/190G/234Y/297W/346A, 174D/215N/234Y/297W/346A,174D/215N/255R/297W/346A, 174D/282K/297W/346A, 190G/255R/282K/346A,190G/297W/346A, 203L/296L, 203L/343R/346H, 203L/343R, 203L/346H,215H/255R/297W/346A, 215N/234Y/297W/346A, 215N/255R/297W/346A,215N/297W, 215N/297W/346A, 215N/346A, 234Y/255R/346A, 255R/297W/346A,255R/346A, 297W/346A, 343R/346H/373M, and 346A, wherein the positionsare numbered with reference to SEQ ID NO:2. In some embodiments, theengineered transglutaminase polypeptides comprise one or moresubstitutions selected from S48K/Y70L/G203L/R254Q/G296L/N343R,S48K/Q170K/G203L, S48K/Q170K/G203L/R254Q/G296L/E346H,S48K/Q170K/G203L/R254Q/G296L/E346H/K373M,S48K/Q170K/G203L/R254Q/E346H/K373M, S48K/Q170K/G203L/R254Q/E346H,S48K/Q170K/G203L/G296L/N343R/E346H, S48K/Q170K/G203L/G296L/E346H/K373M,S48K/Q170K/G203L/N343R/E346H, S48K/Q170K/G203L/E346H/K373M,S48K/Q170K/G203L/E346H, S48K/Q170K/G203L/K373M, S48K/Q170K/R254Q,S48K/Q170K/G296L, S48K/Q170K/G296L/N343R/E346H, S48K/Q170K/N343R/E346H,S48K/G203L, S48K/G203L/R254Q/G296L, S48K/G203L/R254Q/G296L/N343R/K373M,S48K/G203L/R254Q/G296L/E346H/K373M, S48K/G203L/R254Q/E346H,S48K/G203L/R254Q/E346H/K373M, S48K/G203L/G296L/N343R/E346H/K373M,S48K/G203L/G296L/N343R/K373M, S48K/G203L/G296L/E346H,S48K/G203L/G296L/E346H/K373M, S48K/G203L/N343R/E346H,S48K/G203L/N343R/E346H/K373M, S48K/G203L/E346H, S48K/G203L/E346H/K373M,S48K/R254Q/E346H, S48K/N343R/E346H, S48V, S48V/R67E/Y70G,S48V/R67E/Y70G/R181K/G203V/S256G, S48V/R67E/Y70G/R181K/S256G/S345E,S48V/R67E/Y70G/R181K/G296R/S345E/K373V,S48V/R67E/Y70G/G203V/S256G/G296R/S345E, S48V/R67E/Y70G/G203V/S345E,S48V/R67E/Y70G/S256G/G296R/S345E/K373V,S48V/R67E/Y70N/G203V/S256G/S345E/G354H/K373L, S48V/R67E/Y70N/S256G,548V/R67E/G203V/S256G/G296R/K373V, S48V/R67E/G203V/S256G/S345E,S48K/Y70D/Q170K/G203L, S48V/Y70G/G203V/S256G/S345E/K373V,S48V/Y70N/G203V/S256G/S345E, S48V/Y70N/G203V/K373V, S48V/R181K,S48V/R181K/G203V/S256G/S345E, S48V/R181K/G203V/S345E,S48V/R181K/S256G/G296R/S345E, S48V/R181K/G296R, S48V/R181K/G296R/S345E,S48V/G203V, S48V/G203V/S256G, S48V/G203V/S256G/G296R/S345E,S48V/G203V/S345E, S48K/R254Q/G296L, S48V/S256G, S48V/S256G/G296R,S48V/S256G/G296R/S345E, S48V/G296R/S345E, S48V/G296R/K373V,S48V/S345E/K373L, R67E/S256G, R67E/G296R/S345E,P68A/E74T/S190G/P215N/E346A, P68A/F136Y/P215N/S255R/R282K/F297W/E346A,P68A/F136Y/P215N/F297W/E346A, P68A/F136Y/H234Y,P68A/V158I/E174D/H234Y/R282K/F297W/E346A, P68A/V158I/P215N/F297W/E346A,P68A/P215N/F297W/E346A, P68A/H234Y, P68A/R282K/F297W/E346A,P68A/F297W/E346A, E74T/F136Y/E174D/R282K/E346A,E74T/F136Y/E174D/F297W/E346A, E74T/F136Y/E346A, E74T/V158I/S255R/F297W,E74T/S255R/E346A, E74T/E346A, F136Y/V158I/S190G/P215N/S255R/F297W/E346A,F136Y/V158I/P215N/F297W/E346A,F136Y/E174D/P215N/S255R/R282K/F297W/E346A,F136Y/S190G/P215N/F297W/E346A, F136Y/P215N/H234Y/R282K/F297W,F136Y/P215N/H234Y/F297W/E346A, F136Y/P215N/F297W, F136Y/F297W/E346A,V158I/P215N/S255R/E346A, V158I/P215N/E346A,Q170K/G203L/R254Q/G296L/N343R/E346H, Q170K/G203L/R254Q/N343R/K373M,Q170K/G203L/N343R/E346H, E174D/S190G/H234Y/F297W/E346A,E174D/P215N/H234Y/F297W/E346A, E174D/P215N/S255R/F297W/E346A,E174D/R282K/F297W/E346A, S190G/S255R/R282K/E346A, S190G/F297W/E346A,G203L/G296L, G203L/N343R/E346H, G203L/N343R, G203L/E346H,P215H/S255R/F297W/E346A, P215N/H234Y/F297W/E346A,P215N/S255R/F297W/E346A, P215N/F297W, P215N/F297W/E346A, P215N/E346A,H234Y/S255R/E346A, S255R/F297W/E346A, S255R/E346A, F297W/E346A,N343R/E346H/K373M, and E346A, wherein the positions are numbered withreference to SEQ ID NO:2.

In some embodiments, the engineered transglutaminase polypeptidescomprise substitutions at one or more positions selected from33/67/70/181/203/256/296/373, 36/48/203/254/346,48/67/70/181/203/256/296/373, 48/67/70/203/256/296/373,48/67/181/203/256/296/373, 48/67/181/203/256/373, 48/67/181/256/296,48/67/203/256/296/373/378, 48/67/203/256/373, 48/67/203/296/373,48/67/256/296/373, 48/70/181/203/256/296/373, 48/70/181/203/256/373,48/70/181/203/296/373, 48/70/203/256/296/373, 48/70/203/256/373,48/70/203/296, 48/70/203/296/373, 48/70/203/373, 48/70/256/296/373,48/70/296/373, 48/176/203/254/346/373, 48/181/203/256/296/373,48/181/203/256/373, 48/181/203/296, 48/181/203/373, 48/181/256/296/373,48/203/254, 48/203/254/343, 48/203/254/343/346/373,48/203/254/343/355/373, 48/203/254/343/373, 48/203/254/346/373,48/203/254/373, 48/203/256/296, 48/203/256/296/373, 48/203/256/373,48/203/296/373, 48/203/296/373/374, 48/203/343/373, 48/203/373, 48/254,48/254/343/346/373, 48/254/343/373, 48/254/346/373, 48/254/373,48/256/296/373, 48/256/373, 48/373, 67/70/181/203/256/296/373,67/70/181/256/296/373, 67/70/181/373, 67/181/203/256/296,67/181/203/256/296/373, 67/181/203/256/373, 67/203/256/296/373,67/256/296/373, 70/181/203/256/296/373, 70/181/203/296/373, 70/203,70/203/256/296/373, 70/203/256/373, 70/203/296/373,74/136/215/234/282/297/346, 74/136/215/234/282/346, 74/136/215/234/297,74/136/215/234/297/343/346, 74/136/215/234/297/346, 74/136/215/234/346,74/136/215/282/297/346, 74/136/215/282/346, 74/136/215/297/346,74/136/215/346, 74/136/234/282/297/346, 74/136/234/346,74/136/282/297/346, 74/215, 74/215/234/282/297/346, 74/215/282/297/346,74/215/346, 136/215/234/282/297/346, 136/215/282/297,136/215/282/297/346, 136/215/282/346, 136/215/297/346, 136/215/346,136/234/297, 136/234/297/346, 136/234/346, 136/282/297, 181/203/256,181/203/256/296, 181/203/256/296/373, 181/203/256/373, 181/203/296/373,181/203/373, 181/256/296/373, 181/296, 203/224/254/373, 203/254,203/254/343/346/373, 203/254/343/373, 203/254/346, 203/254/346/373,203/254/373, 203/346/373, 203/373, 203/209/256/373, 203/256,203/256/296, 203/256/296/320/373, 203/256/296/373, 203/256/296/373/386,203/256/373, 203/296/373, 203/373, 215/234/282/297/346, 215/234/282/346,215/234/346, 234/282/346, 254, 254/346, 254/346/373, 254/373, 256/296,256/296/373, 256/373, 282/297/346, 343/373, and 373, wherein thepositions are numbered with reference to SEQ ID NO:2. In someembodiments, the engineered transglutaminase polypeptides comprise oneor more substitutions selected from33D/67E/70G/181K/203V/256G/296R/373V, 36E/48K/203L/254Q/346H,48K/176T/203L/254Q/346H/373M, 48K/203L/254Q, 48K/203L/254Q/343R,48K/203L/254Q/343R/346H/373M, 48K/203L/254Q/343R/355T/373M,48K/203L/254Q/343R/373M, 48K/203L/254Q/346D/373M, 48K/203L/254Q/373M,48K/203L/343R/373M, 48K/203L/373M, 48K/254Q, 48K/254Q/343R/346H/373M,48K/254Q/343R/373M, 48K/254Q/346H/373M, 48K/254Q/373M,48V/67E/70G/181K/203V/256G/296R/373V, 48V/67E/70G/203V/256G/296R/373V,48V/67E/181K/203V/256G/296R/373V, 48V/67E/181K/203V/256G/373V,48V/67E/181K/256G/296R, 48V/67E/203V/256G/296R/373V/378D,48V/67E/203V/256G/373V, 48V/67E/203V/296R/373V, 48V/67E/256G/296R/373V,48V/70G/181K/203V/256G/296R/373V, 48V/70G/181K/203V/256G/373V,48V/70G/181K/203V/296R/373V, 48V/70G/203V/256G/296R/373V,48V/70G/203V/256G/373V, 48V/70G/203V/296R, 48V/70G/203V/296R/373V,48V/70G/203V/373V, 48V/70G/256G/296R/373V, 48V/70G/296R/373V,48V/181K/203V/256G/296R/373V, 48V/181K/203V/256G/373V,48V/181K/203V/296R, 48V/181K/203V/373V, 48V/181K/256G/296R/373V,48V/203V/256G/296R, 48V/203V/256G/296R/373V, 48V/203V/256G/373V,48V/203V/296R/373V, 48V/203V/296R/373V/374L, 48V/203V/373V,48V/256G/296R/373V, 48V/256G/373V, 48V/373V,67E/70G/181K/203V/256G/296R/373V, 67E/70G/181K/256G/296R/373V,67E/70G/181K/373V, 67E/181K/203V/256G/296R,67E/181K/203V/256G/296R/373V, 67E/181K/203V/256G/373V,67E/203V/256G/296R/373V, 67E/256G/296R/373V,70G/181K/203V/256G/296R/373V, 70G/181K/203V/296R/373V, 70G/203V,70G/203V/256G/296R/373V, 70G/203V/256G/373V, 70G/203V/296R/373V,74T/136Y/215N/234Y/282K/297W/346A, 74T/136Y/215N/234Y/282K/346A,74T/136Y/215N/234Y/297W, 74T/136Y/215N/234Y/297W/343Y/346A,74T/136Y/215N/234Y/297W/346A, 74T/136Y/215N/234Y/346A,74T/136Y/215N/282K/297W/346A, 74T/136Y/215N/282K/346A,74T/136Y/215N/297W/346A, 74T/136Y/215N/346A,74T/136Y/234Y/282K/297W/346A, 74T/136Y/234Y/346A,74T/136Y/282K/297W/346A, 74T/215N, 74T/215N/234Y/282K/297W/346A,74T/215N/282K/297W/346A, 74T/215N/346A, 136Y/215N/234Y/282K/297W/346A,136Y/215N/282K/297W, 136Y/215N/282K/297W/346A, 136Y/215N/282K/346A,136Y/215N/297W/346A, 136Y/215N/346A, 136Y/234Y/297W,136Y/234Y/297W/346A, 136Y/234Y/346A, 136Y/282K/297W, 181K/203V/256G,181K/203V/256G/296R, 181K/203V/256G/296R/373V, 181K/203V/256G/373V,181K/203V/296R/373V, 181K/203V/373V, 181K/256G/296R/373V, 181K/296R,203L/224T/254Q/373M, 203L/254Q, 203L/254Q/343R/346H/373M,203L/254Q/343R/373M, 203L/254Q/346H, 203L/254Q/346H/373M,203L/254Q/373M, 203L/346H/373M, 203L/373M, 203V/209Y/256G/373V,203V/256G, 203V/256G/296R, 203V/256G/296R/320Y/373V,203V/256G/296R/373V, 203V/256G/296R/373V/386Y, 203V/256G/373V,203V/296R/373V, 203V/373V, 215N/234Y/282K/297W/346A,215N/234Y/282K/346A, 215N/234Y/346A, 234Y/282K/346A, 254Q, 254Q/346H,254Q/346H/373M, 254Q/373M, 256G/296R, 256G/296R/373V, 256G/373V,282K/297W/346A, 343R/373M, and 373M/V, wherein the positions arenumbered with reference to SEQ ID NO:2. In some embodiments, theengineered transglutaminase polypeptides comprise one or moresubstitutions selected fromA33D/R67E/Y70G/R181K/G203V/S256G/G296R/K373V,A36E/S48K/G203L/R254Q/E346H, S48K/A176T/G203L/R254Q/E346H/K373M,S48K/G203L/R254Q, S48K/G203L/R254Q/N343R,S48K/G203L/R254Q/N343R/E346H/K373M, S48K/G203L/R254Q/N343R/A355T/K373M,S48K/G203L/R254Q/N343R/K373M, S48K/G203L/R254Q/E346D/K373M,S48K/G203L/R254Q/K373M, S48K/G203L/N343R/K373M, S48K/G203L/K373M,S48K/R254Q, S48K/R254Q/N343R/E346H/K373M, S48K/R254Q/N343R/K373M,S48K/R254Q/E346H/K373M, S48K/R254Q/K373M,S48V/R67E/Y70G/R181K/G203V/S256G/G296R/K373V,S48V/R67E/Y70G/G203V/S256G/G296R/K373V,S48V/R67E/R181K/G203V/S256G/G296R/K373V,S48V/R67E/R181K/G203V/S256G/K373V, S48V/R67E/R181K/S256G/G296R,S48V/R67E/G203V/S256G/G296R/K373V/G378D, S48V/R67E/G203V/S256G/K373V,S48V/R67E/G203V/G296R/K373V, S48V/R67E/S256G/G296R/K373V,S48V/Y70G/R181K/G203V/S256G/G296R/K373V,S48V/Y70G/R181K/G203V/S256G/K373V, S48V/Y70G/R181K/G203V/G296R/K373V,S48V/Y70G/G203V/S256G/G296R/K373V, S48V/Y70G/G203V/S256G/K373V,S48V/Y70G/G203V/G296R, S48V/Y70G/G203V/G296R/K373V,S48V/Y70G/G203V/K373V, S48V/Y70G/S256G/G296R/K373V,S48V/Y70G/G296R/K373V, S48V/R181K/G203V/S256G/G296R/K373V,S48V/R181K/G203V/S256G/K373V, S48V/R181K/G203V/G296R,S48V/R181K/G203V/K373V, S48V/R181K/S256G/G296R/K373V,S48V/G203V/S256G/G296R, S48V/G203V/S256G/G296R/K373V,S48V/G203V/S256G/K373V, S48V/G203V/G296R/K373V,S48V/G203V/G296R/K373V/Q374L, S48V/G203V/K373V, S48V/S256G/G296R/K373V,S48V/S256G/K373V, S48V/K373V, R67E/Y70G/R181K/G203V/S256G/G296R/K373V,R67E/Y70G/R181K/S256G/G296R/K373V, R67E/Y70G/R181K/K373V,R67E/R181K/G203V/S256G/G296R, R67E/R181K/G203V/S256G/G296R/K373V,R67E/R181K/G203V/S256G/K373V, R67E/G203V/S256G/G296R/K373V,R67E/S256G/G296R/K373V, Y70G/R181K/G203V/S256G/G296R/K373V,Y70G/R181K/G203V/G296R/K373V, Y70G/G203V, Y70G/G203V/S256G/G296R/K373V,Y70G/G203V/S256G/K373V, Y70G/G203V/G296R/K373V,E74T/F136Y/P215N/H234Y/R282K/F297W/E346A,E74T/F136Y/P215N/H234Y/R282K/E346A, E74T/F136Y/P215N/H234Y/F297W,E74T/F136Y/P215N/H234Y/F297W/N343Y/E346A,E74T/F136Y/P215N/H234Y/F297W/E346A, E74T/F136Y/P215N/H234Y/E346A,E74T/F136Y/P215N/R282K/F297W/E346A, E74T/F136Y/P215N/R282K/E346A,E74T/F136Y/P215N/F297W/E346A, E74T/F136Y/P215N/E346A,E74T/F136Y/H234Y/R282K/F297W/E346A, E74T/F136Y/H234Y/E346A,E74T/F136Y/R282K/F297W/E346A, E74T/P215N,E74T/P215N/H234Y/R282K/F297W/E346A, E74T/P215N/R282K/F297W/E346A,E74T/P215N/E346A, F136Y/P215N/H234Y/R282K/F297W/E346A,F136Y/P215N/R282K/F297W, F136Y/P215N/R282K/F297W/E346A,F136Y/P215N/R282K/E346A, F136Y/P215N/F297W/E346A, F136Y/P215N/E346A,F136Y/H234Y/F297W, F136Y/H234Y/F297W/E346A, F136Y/H234Y/E346A,F136Y/R282K/F297W, R181K/G203V/S256G, R181K/G203V/S256G/G296R,R181K/G203V/S256G/G296R/K373V, R181K/G203V/S256G/K373V,R181K/G203V/G296R/K373V, R181K/G203V/K373V, R181K/S256G/G296R/K373V,R181K/G296R, G203L/P224T/R254Q/K373M, G203L/R254Q,G203L/R254Q/N343R/E346H/K373M, G203L/R254Q/N343R/K373M,G203L/R254Q/E346H, G203L/R254Q/E346H/K373M, G203L/R254Q/K373M,G203L/E346H/K373M, G203L/K373M, G203V/N209Y/S256G/K373V, G203V/S256G,G203V/S256G/G296R, G203V/S256G/G296R/H320Y/K373V,G203V/S256G/G296R/K373V, G203V/S256G/G296R/K373V/H386Y,G203V/S256G/K373V, G203V/G296R/K373V, G203V/K373V,P215N/H234Y/R282K/F297W/E346A, P215N/H234Y/R282K/E346A,P215N/H234Y/E346A, H234Y/R282K/E346A, R254Q, R254Q/E346H,R254Q/E346H/K373M, R254Q/K373M, S256G/G296R, S256G/G296R/K373V,S256G/K373V, R282K/F297W/E346A, N343R/K373M, and K373M/V, wherein thepositions are numbered with reference to SEQ ID NO:2.

In some embodiments, the engineered transglutaminase polypeptidescomprise substitutions at one or more positions selected from 48/49, 49,50, 50, 331, 291, 292, 330, and 331, wherein the positions are numberedwith reference to SEQ ID NO:34. In some embodiments, the engineeredtransglutaminase polypeptides comprise one or more substitutionsselected from 48S/49W, 49Y, 50A/F/Q/R, 331H/P/V, 291C, 292R, 330H/Y, and331R, wherein the positions are numbered with reference to SEQ ID NO:34.In some embodiments, the engineered transglutaminase polypeptidescomprise one or more substitutions selected from K48S/D49W, D49Y,D50A/F/Q/R, L331H/P/V, T291C, S292R, S330H/Y, and L331R, wherein thepositions are numbered with reference to SEQ ID NO:34.

In some embodiments, the engineered transglutaminase polypeptidescomprise substitutions at one or more positions selected from27/48/67/70/74/234/256/282/346/373,27/48/67/70/136/203/215/256/282/346/373, 27/48/67/70/346/373,27/48/67/74/203/256/346/373, 27/67/234/296/373, 45/287/328/333,45/292/328, 48, 48/284/292/333, 48/287/292/297, 48/287/297/328/333,48/292, 48/292/297, 48/49/50/292/331, 48/49/50/292, 48/49/50/331,48/49/330/331, 48/49/50/349, 48/49/50/291/292/331, 48/49/50/292/331,48/67/70/203/215/234/256/346, 48/67/70/234/256/282/297/346,48/67/70/346, 48/67/74/203/234/256/282/346/373,48/67/74/234/297/346/373, 48/67/74/346, 48/67/203/346/373,48/67/234/256/297/346/373, 48/67/234/256/346/373,48/67/215/282/297/346/373, 48/67/346/373, 48/70/74/297/346/373,48/70/203/215/256/282/346/373, 48/70/215/234/256/346/373,48/74/203/234/256/346/373, 48/74/234/256/297/346/373,48/136/256/346/373, 48/203/234/256/297/346/373, 48/203/234/256/346/373,48/203/234/346/373, 48/203/296/373, 48/215/234/346/373, 48/215/346/373,48/234/256/296/346/373, 48/234/256/346/373, 48/256/373, 49/50/292/331,49/50/292/331/349, 49/50/331, 49/50/331/349, 50,67/70/74/136/203/215/256/346/373, 67/70/74/203/215/234/346/373,67/70/74/215/234/297/346/373, 67/70/74/215/256/373,67/70/136/203/297/346/373, 67/70/203/215/256/346/373, 67/70/203/373,67/70/215, 67/74/136, 67/74/203/234/256, 67/74/215/256/297/346/373,67/74/215/346/373, 67/74/256/346/373, 67/136/203/215/256/346/373,67/136/203/256/346/373, 67/203/234/256/346/373, 67/203/297/346/373,67/215/234/297/346/373, 67/297/346, 70/74/203/215/346/373, 136,136/346/373, 203/234/346, 203/234/346/373, 203/373, 234/282, 287,234/346/373, 287/292, 287/292/295/297, 287/292/297, 287/295/297,287/330/333, 292, 292/297, 292/330/331, 292/330/331, 292/331,292/331/349, 292/349, 295, 295/297/333, 297/328, 297/373, 328/333, 330,330/331, 331, 331/349, 333, 346/373, and 373, wherein the positions arenumbered with reference to SEQ ID NO:256. In some embodiments, theengineered transglutaminase polypeptides comprise one or moresubstitutions selected from27S/48V/67E/70G/74T/234Y/256G/282K/346A/373L,27S/48V/67E/70G/136Y/203V/215H/256G/282K/346A/373V,27S/48V/67E/70G/346A/373L, 27S/48V/67E/74T/203V/256G/346A/373M,27S/67E/234Y/296R/373M, 45S/287S/328E/333P, 45S/292K/328E, 48A,48A/284G/292K/333P, 48A/287S/292K/297Y, 48A/287S/297Y/328E/333P,48A/292K, 48A/292K/297Y, 48S/49G/50A/292R/331P, 48S/49W/50A/292R,48S/49W/50A/331V, 48S/49W/330Y/331V, 48S/49W/50A/349R,48S/49Y/50A/291C/292R/331V, 48S/49Y/50Q/292R/331V,48V/67E/70G/203V/215H/234Y/256G/346A,48V/67E/70G/234Y/256G/282K/297W/346A, 48V/67E/70G/346A,48V/67E/74T/203V/234Y/256G/282K/346A/373M,48V/67E/74T/234Y/297W/346A/373M, 48V/67E/74T/346A,48V/67E/203V/346A/373M, 48V/67E/215H/282K/297W/346A/373M,48V/67E/234Y/256G/297W/346A/373V, 48V/67E/234Y/256G/346A/373M,48V/67E/346A/373M, 48V/70G/74T/297W/346A/373M,48V/70G/203V/215H/256G/282K/346A/373V, 48V/70G/215H/234Y/256G/346A/373M,48V/74T/203V/234Y/256G/346A/373V, 48V/74T/234Y/256G/297W/346A/373V,48V/136Y/256G/346A/373M, 48V/203V/234Y/256G/297W/346A/373V,48V/203V/234Y/256G/346A/373M, 48V/203V/234Y/346A/373M,48V/203V/296R/373M, 48V/215H/234Y/346A/373V, 48V/215H/346A/373M,48V/234Y/256G/296R/346A/373M, 48V/234Y/256G/346A/373M, 48V/256G/373L,49G/50A/292R/331V, 49G/50Q/292R/331V/349R, 49W/50A/331V,49W/50A/331V/349R, 50A, 67E/70G/74T/136Y/203V/215H/256G/346A/373M,67E/70G/74T/203V/215H/234Y/346A/373V,67E/70G/74T/215H/234Y/297W/346A/373L, 67E/70G/74T/215H/256G/373M,67E/70G/136Y/203V/297W/346A/373M, 67E/70G/203V/215H/256G/346A/373L,67E/70G/203V/373M, 67E/70G/215H, 67E/74T/136Y, 67E/74T/203V/234Y/256G,67E/74T/215H/256G/297W/346A/373L, 67E/74T/215H/346A/373V,67E/74T/256G/346A/373M, 67E/136Y/203V/215H/256G/346A/373V,67E/136Y/203V/256G/346A/373M, 67E/203V/234Y/256G/346A/373V,67E/203V/297W/346A/373M, 67E/215H/234Y/297W/346A/373V, 67E/297W/346A,70G/74T/203V/215H/346A/373V, 136Y, 136Y/346A/373M, 203V/234Y/346A,203V/234Y/346A/373V, 203V/373M, 234Y/282K, 234Y/346A/373M, 287S,287S/292K, 287S/292K/295R/297Y, 287S/292K/297Y, 287S/295R/297Y,287S/330G/333P, 292K, 292K/297Y, 292R, 292R/330Y/331P, 292R/330Y/331V,292R/331V, 292R/331V/349R, 292R/349R, 295R, 295R/297Y/333P, 297Y/328E,297W/373M, 328E/333P, 330Y, 330Y/331P, 331V, 331V/349R, 333P, 346A/373V,and 373M/V, wherein the positions are numbered with reference to SEQ IDNO:256. In some embodiments, the engineered transglutaminasepolypeptides comprise one or more substitutions selected fromN27S/K48V/R67E/Y70G/E74T/H234Y/S256G/R282K/H346A/K373L,N27S/K48V/R67E/Y70G/F136Y/L203V/P215H/S256G/R282K/H346A/K373V,N27S/K48V/R67E/Y70G/H346A/K373L,N27S/K48V/R67E/E74T/L203V/S256G/H346A/K373M,N27S/R67E/H234Y/G296R/K373M, A45S/P287S/N328E/A333P, A45S/S292K/N328E,K48A, K48A/R284G/S292K/A333P, K48A/P287S/S292K/F297Y,K48A/P287S/F297Y/N328E/A333P, K48A/S292K, K48A/S292K/F297Y,K48S/D49G/R50A/S292R/L331P, K48S/D49W/R50A/S292R, K48S/D49W/R50A/L331V,K48S/D49W/S330Y/L331V, K48S/D49W/R50A/S349R,K48S/D49Y/R50A/T291C/S292R/L331V, K48S/D49Y/R50Q/S292R/L331V,K48V/R67E/Y70G/L203V/P215H/H234Y/S256G/H346A,K48V/R67E/Y70G/H234Y/S256G/R282K/F297W/H346A, K48V/R67E/Y70G/H346A,K48V/R67E/E74T/L203V/H234Y/S256G/R282K/H346A/K373M,K48V/R67E/E74T/H234Y/F297W/H346A/K373M, K48V/R67E/E74T/H346A,K48V/R67E/L203V/H346A/K373M, K48V/R67E/P215H/R282K/F297W/H346A/K373M,K48V/R67E/H234Y/S256G/F297W/H346A/K373V,K48V/R67E/H234Y/S256G/H346A/K373M, K48V/R67E/H346A/K373M,K48V/Y70G/E74T/F297W/H346A/K373M,K48V/Y70G/L203V/P215H/S256G/R282K/H346A/K373V,K48V/Y70G/P215H/H234Y/S256G/H346A/K373M,K48V/E74T/L203V/H234Y/S256G/H346A/K373V,K48V/E74T/H234Y/S256G/F297W/H346A/K373V, K48V/F136Y/S256G/H346A/K373M,K48V/L203V/H234Y/S256G/F297W/H346A/K373V,K48V/L203V/H234Y/S256G/H346A/K373M, K48V/L203V/H234Y/H346A/K373M,K48V/L203V/G296R/K373M, K48V/P215H/H234Y/H346A/K373V,K48V/P215H/H346A/K373M, K48V/H234Y/S256G/G296R/H346A/K373M,K48V/H234Y/S256G/H346A/K373M, K48V/S256G/K373L, D49G/R50A/S292R/L331V,D49G/R50Q/S292R/L331V/S349R, D49W/R50A/L331V, D49W/R50A/L331V/S349R,R50A, R67E/Y70G/E74T/F136Y/L203V/P215H/S256G/H346A/K373M,R67E/Y70G/E74T/L203V/P215H/H234Y/H346A/K373V,R67E/Y70G/E74T/P215H/H234Y/F297W/H346A/K373L,R67E/Y70G/E74T/P215H/S256G/K373M,R67E/Y70G/F136Y/L203V/F297W/H346A/K373M,R67E/Y70G/L203V/P215H/S256G/H346A/K373L, R67E/Y70G/L203V/K373M,R67E/Y70G/P215H, R67E/E74T/F136Y, R67E/E74T/L203V/H234Y/S256G,R67E/E74T/P215H/S256G/F297W/H346A/K373L, R67E/E74T/P215H/H346A/K373V,R67E/E74T/S256G/H346A/K373M, R67E/F136Y/L203V/P215H/S256G/H346A/K373V,R67E/F136Y/L203V/S256G/H346A/K373M, R67E/L203V/H234Y/S256G/H346A/K373V,R67E/L203V/F297W/H346A/K373M, R67E/P215H/H234Y/F297W/H346A/K373V,R67E/F297W/H346A, Y70G/E74T/L203V/P215H/H346A/K373V, F136Y,F136Y/H346A/K373M, L203V/H234Y/H346A, L203V/H234Y/H346A/K373V,L203V/K373M, H234Y/R282K, H234Y/H346A/K373M, P287S, P287S/S292K,P287S/S292K/E295R/F297Y, P287S/S292K/F297Y, P287S/E295R/F297Y,P287S/S330G/A333P, S292K, S292K/F297Y, S292R, S292R/S330Y/L331P,S292R/S330Y/L331V, S292R/L331V, S292R/L331V/S349R, S292R/S349R, E295R,E295R/F297Y/A333P, F297Y/N328E, F297W/K373M, N328E/A333P, S330Y,S330Y/L331P, L331V, L331V/S349R, A333P, H346A/K373V, and K373M/V,wherein the positions are numbered with reference to SEQ ID NO:256.

The present invention also provides polynucleotides encoding theengineered transglutaminase polypeptides. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression, to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered transglutaminase polypeptides can be introduced intoappropriate host cells to express the corresponding transglutaminasepolypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improvedtransglutaminase enzymes disclosed herein. Thus, having identified aparticular amino acid sequence, those skilled in the art could make anynumber of different nucleic acids by simply modifying the sequence ofone or more codons in a way which does not change the amino acidsequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made by selecting combinations based onthe possible codon choices, and all such variations are to be consideredspecifically disclosed for any polypeptide disclosed herein, includingthe amino acid sequences presented in the Tables in the Examples herein.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells.

In some embodiments, all codons need not be replaced to optimize thecodon usage of the transglutaminase polypeptides since the naturalsequence will comprise preferred codons and because use of preferredcodons may not be required for all amino acid residues. Consequently,codon optimized polynucleotides encoding the transglutaminase enzymesmay contain preferred codons at about 40%, 50%, 60%, 70%, 80%, orgreater than 90% of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a transglutaminase polypeptide with an amino acid sequence thathas at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 2, 6,34, and/or 256. In some embodiments, the polynucleotide comprises anucleotide sequence encoding a transglutaminase polypeptide with anamino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity toSEQ ID NO: 2, 6, 34, and/or 256. In some embodiments, the polynucleotideencodes a transglutaminase amino acid sequence of SEQ ID NO: 2, 6, 34,and/or 256. In some embodiments, the present invention providespolynucleotide sequences having at least about 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequenceidentity to SEQ ID NO: 1, 5, 33, and/or 255. In some embodiments, thepresent invention provides polynucleotide sequences having at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or more sequence identity to SEQ ID NO: 1, 5, 33, and/or 255.

In some embodiments, the isolated polynucleotide encoding an improved Insome embodiments, the polynucleotide comprises a nucleotide sequenceencoding a transglutaminase polypeptide with an amino acid sequence thathas at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 2, 6,34, and/or 256. The polypeptide is manipulated in a variety of ways toprovide for improved activity and/or expression of the polypeptide.Manipulation of the isolated polynucleotide prior to its insertion intoa vector may be desirable or necessary depending on the expressionvector. The techniques for modifying polynucleotides and nucleic acidsequences utilizing recombinant DNA methods are well known in the art.

For example, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Mutagenesis and directed evolutionmethods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548,6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201,6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344,6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160,6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742,6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246,6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855,6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224,6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011,6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603,6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098,6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514,6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922,6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515,7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347,7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170,7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428,7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499,7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674,8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681,8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871,9,593,326, and all related US and non-US counterparts; Ling et al.,Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol.,57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botsteinet al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7[1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene,34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290[1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameriet al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of whichare incorporated herein by reference).

In some embodiments, the variant transglutaminase of the presentinvention further comprise additional sequences that do not alter theencoded activity of the enzyme. For example, in some embodiments, thevariant transglutaminase are linked to an epitope tag or to anothersequence useful in purification.

In some embodiments, the variant transglutaminase polypeptides of thepresent invention are secreted from the host cell in which they areexpressed (e.g., a yeast or filamentous fungal host cell) and areexpressed as a pre-protein including a signal peptide (i.e., an aminoacid sequence linked to the amino terminus of a polypeptide and whichdirects the encoded polypeptide into the cell secretory pathway).

In some embodiments, the signal peptide is an endogenous S. mobaraensistransglutaminase signal peptide. In some additional embodiments, signalpeptides from other S. mobaraensis secreted proteins are used. In someembodiments, other signal peptides find use, depending on the host celland other factors. Effective signal peptide coding regions forfilamentous fungal host cells include, but are not limited to, thesignal peptide coding regions obtained from Aspergillus oryzae TAKAamylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, Humicola lanuginosa lipase, and T. reesei cellobiohydrolaseII. Signal peptide coding regions for bacterial host cells include, butare not limited to the signal peptide coding regions obtained from thegenes for Bacillus NC1B 11837 maltogenic amylase, Bacillusstearothermophilus alpha-amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutralproteases (nprT, nprS, nprM), and Bacillus subtilis prsA. In someadditional embodiments, other signal peptides find use in the presentinvention (See e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137[1993], incorporated herein by reference). Additional useful signalpeptides for yeast host cells include those from the genes forSaccharomyces cerevisiae alpha-factor, Saccharomyces cerevisiae SUC2invertase (See e.g., Taussig and Carlson, Nucl. Acids Res., 11:1943-54[1983]; SwissProt Accession No. P00724; and Romanos et al., Yeast8:423-488 [1992]). In some embodiments, variants of these signalpeptides and other signal peptides find use. Indeed, it is not intendedthat the present invention be limited to any specific signal peptide, asany suitable signal peptide known in the art finds use in the presentinvention.

In some embodiments, the present invention provides polynucleotidesencoding variant transglutaminase polypeptides, and/or biologicallyactive fragments thereof, as described herein. In some embodiments, thepolynucleotide is operably linked to one or more heterologous regulatoryor control sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide. Insome embodiments, expression constructs containing a heterologouspolynucleotide encoding a variant transglutaminase is introduced intoappropriate host cells to express the variant transglutaminase.

Those of ordinary skill in the art understand that due to the degeneracyof the genetic code, a multitude of nucleotide sequences encodingvariant transglutaminase polypeptides of the present invention exist.For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode theamino acid arginine. Thus, at every position in the nucleic acids of theinvention where an arginine is specified by a codon, the codon can bealtered to any of the corresponding codons described above withoutaltering the encoded polypeptide. It is understood that “U” in an RNAsequence corresponds to “T” in a DNA sequence. The inventioncontemplates and provides each and every possible variation of nucleicacid sequence encoding a polypeptide of the invention that could be madeby selecting combinations based on possible codon choices.

As indicated above, DNA sequence encoding a transglutaminase may also bedesigned for high codon usage bias codons (codons that are used athigher frequency in the protein coding regions than other codons thatcode for the same amino acid). The preferred codons may be determined inrelation to codon usage in a single gene, a set of genes of commonfunction or origin, highly expressed genes, the codon frequency in theaggregate protein coding regions of the whole organism, codon frequencyin the aggregate protein coding regions of related organisms, orcombinations thereof. A codon whose frequency increases with the levelof gene expression is typically an optimal codon for expression. Inparticular, a DNA sequence can be optimized for expression in aparticular host organism. A variety of methods are well-known in the artfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis (e.g., using cluster analysis orcorrespondence analysis,) and the effective number of codons used in agene. The data source for obtaining codon usage may rely on anyavailable nucleotide sequence capable of coding for a protein. Thesedata sets include nucleic acid sequences actually known to encodeexpressed proteins (e.g., complete protein coding sequences-CDS),expressed sequence tags (ESTs), or predicted coding regions of genomicsequences, as is well-known in the art. Polynucleotides encoding varianttransglutaminases can be prepared using any suitable methods known inthe art. Typically, oligonucleotides are individually synthesized, thenjoined (e.g., by enzymatic or chemical ligation methods, orpolymerase-mediated methods) to form essentially any desired continuoussequence. In some embodiments, polynucleotides of the present inventionare prepared by chemical synthesis using, any suitable methods known inthe art, including but not limited to automated synthetic methods. Forexample, in the phosphoramidite method, oligonucleotides are synthesized(e.g., in an automatic DNA synthesizer), purified, annealed, ligated andcloned in appropriate vectors. In some embodiments, double stranded DNAfragments are then obtained either by synthesizing the complementarystrand and annealing the strands together under appropriate conditions,or by adding the complementary strand using DNA polymerase with anappropriate primer sequence. There are numerous general and standardtexts that provide methods useful in the present invention are wellknown to those skilled in the art.

The engineered transglutaminases can be obtained by subjecting thepolynucleotide encoding the naturally occurring transglutaminase tomutagenesis and/or directed evolution methods, as discussed above.Mutagenesis may be performed in accordance with any of the techniquesknown in the art, including random and site-specific mutagenesis.Directed evolution can be performed with any of the techniques known inthe art to screen for improved variants including shuffling. Otherdirected evolution procedures that find use include, but are not limitedto staggered extension process (StEP), in vitro recombination, mutagenicPCR, cassette mutagenesis, splicing by overlap extension (SOEing),ProSAR™ directed evolution methods, etc., as well as any other suitablemethods.

The clones obtained following mutagenesis treatment are screened forengineered transglutaminases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofproduct formation. Where an improved enzyme property desired is thermalstability, enzyme activity may be measured after subjecting the enzymepreparations to a defined temperature and measuring the amount of enzymeactivity remaining after heat treatments. Clones containing apolynucleotide encoding a transglutaminase are then isolated, sequencedto identify the nucleotide sequence changes (if any), and used toexpress the enzyme in a host cell.

When the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis (e.g., using theclassical phosphoramidite method described by Beaucage et al., Tet.Lett., 22:1859-69 [1981], or the method described by Matthes et al.,EMBO J., 3:801-05 [1984], as it is typically practiced in automatedsynthetic methods). According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors. In addition, essentially any nucleic acid can be obtained fromany of a variety of commercial sources (e.g., The Midland CertifiedReagent Company, Midland, Tex., The Great American Gene Company, Ramona,Calif., ExpressGen Inc. Chicago, Ill., Operon Technologies Inc.,Alameda, Calif., and many others).

The present invention also provides recombinant constructs comprising asequence encoding at least one variant transglutaminase, as providedherein. In some embodiments, the present invention provides anexpression vector comprising a variant transglutaminase polynucleotideoperably linked to a heterologous promoter. In some embodiments,expression vectors of the present invention are used to transformappropriate host cells to permit the host cells to express the varianttransglutaminase protein. Methods for recombinant expression of proteinsin fungi and other organisms are well known in the art, and a number ofexpression vectors are available or can be constructed using routinemethods. In some embodiments, nucleic acid constructs of the presentinvention comprise a vector, such as, a plasmid, a cosmid, a phage, avirus, a bacterial artificial chromosome (BAC), a yeast artificialchromosome (YAC), and the like, into which a nucleic acid sequence ofthe invention has been inserted. In some embodiments, polynucleotides ofthe present invention are incorporated into any one of a variety ofexpression vectors suitable for expressing variant transglutaminasepolypeptide(s). Suitable vectors include, but are not limited tochromosomal, nonchromosomal and synthetic DNA sequences (e.g.,derivatives of SV40), as well as bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, pseudorabies, adenovirus, adeno-associated virus, retroviruses,and many others. Any suitable vector that transduces genetic materialinto a cell, and, if replication is desired, which is replicable andviable in the relevant host finds use in the present invention.

In some embodiments, the construct further comprises regulatorysequences, including but not limited to a promoter, operably linked tothe protein encoding sequence. Large numbers of suitable vectors andpromoters are known to those of skill in the art. Indeed, in someembodiments, in order to obtain high levels of expression in aparticular host it is often useful to express the varianttransglutaminases of the present invention under the control of aheterologous promoter. In some embodiments, a promoter sequence isoperably linked to the 5′ region of the variant transglutaminase codingsequence using any suitable method known in the art. Examples of usefulpromoters for expression of variant transglutaminases include, but arenot limited to promoters from fungi. In some embodiments, a promotersequence that drives expression of a gene other than a transglutaminasegene in a fungal strain finds use. As a non-limiting example, a fungalpromoter from a gene encoding an endoglucanase may be used. In someembodiments, a promoter sequence that drives the expression of atransglutaminase gene in a fungal strain other than the fungal strainfrom which the transglutaminases were derived finds use. Examples ofother suitable promoters useful for directing the transcription of thenucleotide constructs of the present invention in a filamentous fungalhost cell include, but are not limited to promoters obtained from thegenes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-likeprotease (See e.g., WO 96/00787, incorporated herein by reference), aswell as the NA2-tpi promoter (a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase), promoters such as cbh1, cbh2, egl1, egl2,pepA, hfb1, hfb2, xyn1, amy, and glaA (See e.g., Nunberg et al., Mol.Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBO J., 3:1581-85 [1984];and European Patent Appln. 137280, all of which are incorporated hereinby reference), and mutant, truncated, and hybrid promoters thereof.

In yeast host cells, useful promoters include, but are not limited tothose from the genes for Saccharomyces cerevisiae enolase (eno-1),Saccharomyces cerevisiae galactokinase (gall), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and S. cerevisiae 3-phosphoglycerate kinase. Additionaluseful promoters useful for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992], incorporated herein byreference). In addition, promoters associated with chitinase productionin fungi find use in the present invention (See e.g., Blaiseau andLafay, Gene 120243-248 [1992]; and Limon et al., Curr. Genet., 28:478-83[1995], both of which are incorporated herein by reference).

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include butare not limited to the promoters obtained from the E. coli lac operon,E. coli trp operon, bacteriophage lambda, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc.Natl. Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter(See e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 [1983]).

In some embodiments, cloned variant transglutaminases of the presentinvention also have a suitable transcription terminator sequence, asequence recognized by a host cell to terminate transcription. Theterminator sequence is operably linked to the 3′ terminus of the nucleicacid sequence encoding the polypeptide. Any terminator that isfunctional in the host cell of choice finds use in the presentinvention. Exemplary transcription terminators for filamentous fungalhost cells include, but are not limited to those obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease (Seee.g., U.S. Pat. No. 7,399,627, incorporated herein by reference). Insome embodiments, exemplary terminators for yeast host cells includethose obtained from the genes for Saccharomyces cerevisiae enolase,Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are well-known to those skilled in theart (See e.g., Romanos et al., Yeast 8:423-88 [1992]).

In some embodiments, a suitable leader sequence is part of a clonedvariant transglutaminase sequence, which is a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′ terminus of the nucleic acidsequence encoding the polypeptide. Any leader sequence that isfunctional in the host cell of choice finds use in the presentinvention. Exemplary leaders for filamentous fungal host cells include,but are not limited to those obtained from the genes for Aspergillusoryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.Suitable leaders for yeast host cells include, but are not limited tothose obtained from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase,Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP).

In some embodiments, the sequences of the present invention alsocomprise a polyadenylation sequence, which is a sequence operably linkedto the 3′ terminus of the nucleic acid sequence and which, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice finds use in the presentinvention. Exemplary polyadenylation sequences for filamentous fungalhost cells include, but are not limited to those obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Fusarium oxysporumtrypsin-like protease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are known in the art (Seee.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

In some embodiments, the control sequence comprises a signal peptidecoding region encoding an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. The foreign signalpeptide coding region may be required where the coding sequence does notnaturally contain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cellsinclude, but are not limited to the signal peptide coding regionsobtained from the genes for Bacillus NC1B 11837 maltogenic amylase,Bacillus stearothermophilus alpha-amylase, Bacillus licheniformissubtilisin, Bacillus licheniformis beta-lactamase, Bacillusstearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillussubtilis prsA. Further signal peptides are known in the art (See e.g.,Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]).

Effective signal peptide coding regions for filamentous fungal hostcells include, but are not limited to the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase.

Useful signal peptides for yeast host cells include, but are not limitedto genes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Other useful signal peptide coding regions areknown in the art (See e.g., Romanos et al., [1992], supra).

In some embodiments, the control sequence comprises a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is known as aproenzyme or propolypeptide (or a zymogen in some cases). Apropolypeptide is generally inactive and can be converted to a matureactive transglutaminase polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila lactase (See e.g., WO95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

In some embodiments, regulatory sequences are also used to allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude, but are not limited to the lac, tac, and trp operator systems.In yeast host cells, suitable regulatory systems include, as examples,the ADH2 system or GAL1 system. In filamentous fungi, suitableregulatory sequences include the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the transglutaminasepolypeptide of the present invention would be operably linked with theregulatory sequence.

Thus, in additional embodiments, the present invention providesrecombinant expression vectors comprising a polynucleotide encoding anengineered transglutaminase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. In some embodiments, the various nucleic acidand control sequences described above are joined together to produce arecombinant expression vector that may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, insome embodiments, the nucleic acid sequences are expressed by insertingthe nucleic acid sequence or a nucleic acid construct comprising thesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector comprises any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. In some embodiments, the vectors are linear or closedcircular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome). In some embodiments, thevector contains any means for assuring self-replication. Alternatively,in some other embodiments, upon being introduced into the host cell, thevector is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, inadditional embodiments, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon find use.

In some embodiments, the expression vector of the present inventioncontains one or more selectable markers, which permit easy selection oftransformed cells. A “selectable marker” is a gene, the product of whichprovides for biocide or viral resistance, resistance to antimicrobialsor heavy metals, prototrophy to auxotrophs, and the like. Any suitableselectable markers for use in a filamentous fungal host cell find use inthe present invention, including, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Additional markers useful in host cells such as Aspergillus, include butare not limited to the amdS and pyrG genes of Aspergillus nidulans orAspergillus oryzae, and the bar gene of Streptomyces hygroscopicus.Suitable markers for yeast host cells include, but are not limited toADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Examples of bacterialselectable markers include, but are not limited to the dal genes fromBacillus subtilis or Bacillus licheniformis, or markers, which conferantibiotic resistance such as ampicillin, kanamycin, chloramphenicol,and or tetracycline resistance.

In some embodiments, the expression vectors of the present inventioncontain an element(s) that permits integration of the vector into thehost cell's genome or autonomous replication of the vector in the cellindependent of the genome. In some embodiments involving integrationinto the host cell genome, the vectors rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector forintegration of the vector into the genome by homologous or nonhomologousrecombination.

In some alternative embodiments, the expression vectors containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements preferably contain a sufficient number ofnucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000base pairs, and most preferably 800 to 10,000 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAM□1 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes its functioningtemperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl.Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence ofthe present invention is inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors include,but are not limited to the p3×FLAG™™ expression vectors (Sigma-AldrichChemicals), which include a CMV promoter and hGH polyadenylation sitefor expression in mammalian host cells and a pBR322 origin ofreplication and ampicillin resistance markers for amplification in E.coli. Other suitable expression vectors include, but are not limited topBluescriptII SK(−) and pBK-CMV (Stratagene), and plasmids derived frompBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly(See e.g., Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector comprising a sequence encoding atleast one variant transglutaminase is transformed into a host cell inorder to allow propagation of the vector and expression of the varianttransglutaminase(s). In some embodiments, the variant transglutaminasesare post-translationally modified to remove the signal peptide and insome cases may be cleaved after secretion. In some embodiments, thetransformed host cell described above is cultured in a suitable nutrientmedium under conditions permitting the expression of the varianttransglutaminase(s). Any suitable medium useful for culturing the hostcells finds use in the present invention, including, but not limited tominimal or complex media containing appropriate supplements. In someembodiments, host cells are grown in HTP media. Suitable media areavailable from various commercial suppliers or may be prepared accordingto published recipes (e.g., in catalogues of the American Type CultureCollection).

In another aspect, the present invention provides host cells comprisinga polynucleotide encoding an improved transglutaminase polypeptideprovided herein, the polynucleotide being operatively linked to one ormore control sequences for expression of the transglutaminase enzyme inthe host cell. Host cells for use in expressing the transglutaminasepolypeptides encoded by the expression vectors of the present inventionare well known in the art and include but are not limited to, bacterialcells, such as E. coli, Bacillus megaterium, Lactobacillus kefir,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture media and growthconditions for the above-described host cells are well known in the art.

Polynucleotides for expression of the transglutaminase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells areknown to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitableeukaryotic host cells include, but are not limited to, fungal cells,algal cells, insect cells, and plant cells. Suitable fungal host cellsinclude, but are not limited to, Ascomycota, Basidiomycota,Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, thefungal host cells are yeast cells and filamentous fungal cells. Thefilamentous fungal host cells of the present invention include allfilamentous forms of the subdivision Eumycotina and Oomycota.Filamentous fungi are characterized by a vegetative mycelium with a cellwall composed of chitin, cellulose and other complex polysaccharides.The filamentous fungal host cells of the present invention aremorphologically distinct from yeast.

In some embodiments of the present invention, the filamentous fungalhost cells are of any suitable genus and species, including, but notlimited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora,Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces,Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium,Sporotrichum, Talaromyces, Thennoascus, Thielavia, Trametes,Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/orteleomorphs, or anamorphs, and synonyms, basionyms, or taxonomicequivalents thereof.

In some embodiments of the present invention, the host cell is a yeastcell, including but not limited to cells of Candida, Hansenula,Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowiaspecies. In some embodiments of the present invention, the yeast cell isHansenula polymorpha, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis,Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thennotolerans, Pichiasalictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichiamethanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, orYarrowia lipolytica.

In some embodiments of the invention, the host cell is an algal cellsuch as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp.ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitableprokaryotic cells include, but are not limited to Gram-positive,Gram-negative and Gram-variable bacterial cells. Any suitable bacterialorganism finds use in the present invention, including but not limitedto Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter,Acidothennus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium,Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter,Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia,Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium,Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus,Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella,Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula,Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella,Yersinia and Zymomonas. In some embodiments, the host cell is a speciesof Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium,Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium,Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus,Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella,Streptococcus, Streptomyces, or Zymomonas. In some embodiments, thebacterial host strain is non-pathogenic to humans. In some embodimentsthe bacterial host strain is an industrial strain. Numerous bacterialindustrial strains are known and suitable in the present invention. Insome embodiments of the present invention, the bacterial host cell is anAgrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A.rubi). In some embodiments of the present invention, the bacterial hostcell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A.globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A.paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A.ureafaciens). In some embodiments of the present invention, thebacterial host cell is a Bacillus species (e.g., B. thuringensis, B.anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B.pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius,B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, andB. amyloliquefaciens). In some embodiments, the host cell is anindustrial Bacillus strain including but not limited to B. subtilis, B.pumilus, B. licheniformis, B. megaterium, B. clausii, B.stearothermophilus, or B. amyloliquefaciens. In some embodiments, theBacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B.stearothermophilus, and/or B. amyloliquefaciens. In some embodiments,the bacterial host cell is a Clostridium species (e.g., C.acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum,C. perfringens, and C. beijerinckii). In some embodiments, the bacterialhost cell is a Corynebacterium species (e.g., C. glutamicum and C.acetoacidophilum). In some embodiments the bacterial host cell is anEscherichia species (e.g., E. coli). In some embodiments, the host cellis Escherichia coli W3110. In some embodiments, the bacterial host cellis an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E.herbicola, E. punctata, and E. terreus). In some embodiments, thebacterial host cell is a Pantoea species (e.g., P. citrea, and P.agglomerans). In some embodiments the bacterial host cell is aPseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, andP. sp. D-0l 10). In some embodiments, the bacterial host cell is aStreptococcus species (e.g., S. equisimiles, S. pyogenes, and S.uberis). In some embodiments, the bacterial host cell is a Streptomycesspecies (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S.coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, andS. lividans). In some embodiments, the bacterial host cell is aZymomonas species (e.g., Z. mobilis, and Z lipolytica).

Many prokaryotic and eukaryotic strains that find use in the presentinvention are readily available to the public from a number of culturecollections such as American Type Culture Collection (ATCC), DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSM), CentraalbureauVoor Schimmelcultures (CBS), and Agricultural Research Service PatentCulture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to havecharacteristics that improve protein secretion, protein stability and/orother properties desirable for expression and/or secretion of a protein.Genetic modification can be achieved by genetic engineering techniquesand/or classical microbiological techniques (e.g., chemical or UVmutagenesis and subsequent selection). Indeed, in some embodiments,combinations of recombinant modification and classical selectiontechniques are used to produce the host cells. Using recombinanttechnology, nucleic acid molecules can be introduced, deleted, inhibitedor modified, in a manner that results in increased yields oftransglutaminase variant(s) within the host cell and/or in the culturemedium. For example, knockout of Alp1 function results in a cell that isprotease deficient, and knockout of pyr5 function results in a cell witha pyrimidine deficient phenotype. In one genetic engineering approach,homologous recombination is used to induce targeted gene modificationsby specifically targeting a gene in vivo to suppress expression of theencoded protein. In alternative approaches, siRNA, antisense and/orribozyme technology find use in inhibiting gene expression. A variety ofmethods are known in the art for reducing expression of protein incells, including, but not limited to deletion of all or part of the geneencoding the protein and site-specific mutagenesis to disrupt expressionor activity of the gene product. (See e.g., Chaveroche et al., Nucl.Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant MicrobeInteract., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett.,30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352[2004]; and You et al., Arch. Micriobiol., 191:615-622 [2009], all ofwhich are incorporated by reference herein). Random mutagenesis,followed by screening for desired mutations also finds use (See e.g.,Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon etal., Eukary Cell 2:247-55 [2003], both of which are incorporated byreference).

Introduction of a vector or DNA construct into a host cell can beaccomplished using any suitable method known in the art, including butnot limited to calcium phosphate transfection, DEAE-dextran mediatedtransfection, PEG-mediated transformation, electroporation, or othercommon techniques known in the art. In some embodiments, the Escherichiacoli expression vector pCK100900i (See, US Pat. Appln. Publn.2006/0195947, which is hereby incorporated by reference herein) findsuse.

In some embodiments, the engineered host cells (i.e., “recombinant hostcells”) of the present invention are cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants, or amplifying the transglutaminase polynucleotide.Culture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and arewell-known to those skilled in the art. As noted, many standardreferences and texts are available for the culture and production ofmany cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin.

In some embodiments, cells expressing the variant transglutaminasepolypeptides of the invention are grown under batch or continuousfermentations conditions. Classical “batch fermentation” is a closedsystem, wherein the compositions of the medium is set at the beginningof the fermentation and is not subject to artificial alternations duringthe fermentation. A variation of the batch system is a “fed-batchfermentation” which also finds use in the present invention. In thisvariation, the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression islikely to inhibit the metabolism of the cells and where it is desirableto have limited amounts of substrate in the medium. Batch and fed-batchfermentations are common and well known in the art. “Continuousfermentation” is an open system where a defined fermentation medium isadded continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation systemsstrive to maintain steady state growth conditions. Methods formodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology.

In some embodiments of the present invention, cell-freetranscription/translation systems find use in producing varianttransglutaminase(s). Several systems are commercially available and themethods are well-known to those skilled in the art.

The present invention provides methods of making varianttransglutaminase polypeptides or biologically active fragments thereof.In some embodiments, the method comprises: providing a host celltransformed with a polynucleotide encoding an amino acid sequence thatcomprises at least about 70% (or at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%) sequence identity to SEQ ID NO: 2, 6, 34, and/or 256, andcomprising at least one mutation as provided herein; culturing thetransformed host cell in a culture medium under conditions in which thehost cell expresses the encoded variant transglutaminase polypeptide;and optionally recovering or isolating the expressed varianttransglutaminase polypeptide, and/or recovering or isolating the culturemedium containing the expressed variant transglutaminase polypeptide. Insome embodiments, the methods further provide optionally lysing thetransformed host cells after expressing the encoded transglutaminasepolypeptide and optionally recovering and/or isolating the expressedvariant transglutaminase polypeptide from the cell lysate. The presentinvention further provides methods of making a variant transglutaminasepolypeptide comprising cultivating a host cell transformed with avariant transglutaminase polypeptide under conditions suitable for theproduction of the variant transglutaminase polypeptide and recoveringthe variant transglutaminase polypeptide. Typically, recovery orisolation of the transglutaminase polypeptide is from the host cellculture medium, the host cell or both, using protein recovery techniquesthat are well known in the art, including those described herein. Insome embodiments, host cells are harvested by centrifugation, disruptedby physical or chemical means, and the resulting crude extract retainedfor further purification. Microbial cells employed in expression ofproteins can be disrupted by any convenient method, including, but notlimited to freeze-thaw cycling, sonication, mechanical disruption,and/or use of cell lysing agents, as well as many other suitable methodswell known to those skilled in the art.

Engineered transglutaminase enzymes expressed in a host cell can berecovered from the cells and/or the culture medium using any one or moreof the techniques known in the art for protein purification, including,among others, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™(Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide isrecovered/isolated and optionally purified by any of a number of methodsknown in the art. For example, in some embodiments, the polypeptide isisolated from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, chromatography (e.g., ion exchange, affinity,hydrophobic interaction, chromatofocusing, and size exclusion), orprecipitation. In some embodiments, protein refolding steps are used, asdesired, in completing the configuration of the mature protein. Inaddition, in some embodiments, high performance liquid chromatography(HPLC) is employed in the final purification steps. For example, in someembodiments, methods known in the art, find use in the present invention(See e.g., Parry et al., Biochem. J., 353:117 [2001]; and Hong et al.,Appl. Microbiol. Biotechnol., 73:1331 [2007], both of which areincorporated herein by reference). Indeed, any suitable purificationmethods known in the art find use in the present invention.

Chromatographic techniques for isolation of the transglutaminasepolypeptide include, but are not limited to reverse phase chromatographyhigh performance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,are known to those skilled in the art.

In some embodiments, affinity techniques find use in isolating theimproved transglutaminase enzymes. For affinity chromatographypurification, any antibody which specifically binds the transglutaminasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with the transglutaminase. The transglutaminasepolypeptide may be attached to a suitable carrier, such as BSA, by meansof a side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

In some embodiments, the transglutaminase variants are prepared and usedin the form of cells expressing the enzymes, as crude extracts, or asisolated or purified preparations. In some embodiments, thetransglutaminase variants are prepared as lyophilisates, in powder form(e.g., acetone powders), or prepared as enzyme solutions. In someembodiments, the transglutaminase variants are in the form ofsubstantially pure preparations.

In some embodiments, the transglutaminase polypeptides are attached toany suitable solid substrate. Solid substrates include but are notlimited to a solid phase, surface, and/or membrane. Solid supportsinclude, but are not limited to organic polymers such as polystyrene,polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, andpolyacrylamide, as well as co-polymers and grafts thereof. A solidsupport can also be inorganic, such as glass, silica, controlled poreglass (CPG), reverse phase silica or metal, such as gold or platinum.The configuration of the substrate can be in the form of beads, spheres,particles, granules, a gel, a membrane or a surface. Surfaces can beplanar, substantially planar, or non-planar. Solid supports can beporous or non-porous, and can have swelling or non-swellingcharacteristics. A solid support can be configured in the form of awell, depression, or other container, vessel, feature, or location. Aplurality of supports can be configured on an array at variouslocations, addressable for robotic delivery of reagents, or by detectionmethods and/or instruments.

In some embodiments, immunological methods are used to purifytransglutaminase variants. In one approach, antibody raised against avariant transglutaminase polypeptide (e.g., against a polypeptidecomprising any of SEQ ID NO: 2, 6, 34, and/or 256 and/or an immunogenicfragment thereof) using conventional methods is immobilized on beads,mixed with cell culture media under conditions in which the varianttransglutaminase is bound, and precipitated. In a related approach,immunochromatography finds use.

In some embodiments, the variant transglutaminases are expressed as afusion protein including a non-enzyme portion. In some embodiments, thevariant transglutaminase sequence is fused to a purificationfacilitating domain. As used herein, the term “purification facilitatingdomain” refers to a domain that mediates purification of the polypeptideto which it is fused. Suitable purification domains include, but are notlimited to metal chelating peptides, histidine-tryptophan modules thatallow purification on immobilized metals, a sequence which bindsglutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to anepitope derived from the influenza hemagglutinin protein; See e.g.,Wilson et al., Cell 37:767 [1984]), maltose binding protein sequences,the FLAG epitope utilized in the FLAGS extension/affinity purificationsystem (e.g., the system available from Immunex Corp), and the like. Oneexpression vector contemplated for use in the compositions and methodsdescribed herein provides for expression of a fusion protein comprisinga polypeptide of the invention fused to a polyhistidine region separatedby an enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography;See e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) while theenterokinase cleavage site provides a means for separating the varianttransglutaminase polypeptide from the fusion protein. pGEX vectors(Promega) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to ligand-agarose beads (e.g., glutathione-agarose in thecase of GST-fusions) followed by elution in the presence of free ligand.

EXPERIMENTAL

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); RT (room temperature); CDS (coding sequence);DNA (deoxyribonucleic acid); RNA (ribonucleic acid); aa (amino acid); TB(Terrific Broth; 12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/Lglycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO₄); LB (LuriaBertani broth); CAM (chloramphenicol); PMBS (polymyxin B sulfate); IPTG(isopropyl thiogalactoside); PEG (polyethylene glycol); TFA(trifluoroacetic acid); CHES (2-cyclohexylamino)ethanesulfonic acid;acetonitrile (MeCN); dimethylsulfoxide (DMSO); dimethylacetamide (DMAc);HPLC (high performance liquid chromatography); UPLC (ultra performanceliquid chromatography); FIOPC (fold improvement over positive control);HTP (high throughput); MWD (multiple wavelength detector); UV(ultraviolet); Codexis (Codexis, Inc., Redwood City, Calif.);Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Millipore (Millipore,Corp., Billerica Mass.); Difco (Difco Laboratories, BD DiagnosticSystems, Detroit, Mich.); GeneOracle (GeneOracle, Santa Clara, Calif.);Boca Scientific (Boca Scientific, Ind., Boca Raton, Fla.); Pall (PallCorporation, Port Washington, N.Y.); Vivaproducts (Vivaproducts, Inc.,Littleton, Mass.); Thermotron (Thermotron, Inc., Holland, Mich.); Infors(Infors USA, Inc., Annapolis Junction, Md.); Genetix (Genetic USA Inc.,Beaverton, Oreg.); Daicel (Daicel, West Chester, Pa.); Genetix (GenetixUSA, Inc., Beaverton, Oreg.); Molecular Devices (Molecular Devices, LLC,Sunnyvale, Calif.); Applied Biosystems (Applied Biosystems, part of LifeTechnologies, Corp., Grand Island, N.Y.); Life Technologies (LifeTechnologies, Corp., Grand Island, N.Y.); Agilent (Agilent Technologies,Inc., Santa Clara, Calif.); Thermo Scientific (part of Thermo FisherScientific, Waltham, Mass.); (Infors; Infors-HT, Bottmingen/Basel,Switzerland); Corning (Corning, Inc., Palo Alto, Calif.); and Bio-Rad(Bio-Rad Laboratories, Hercules, Calif.); Microfluidics (MicrofluidicsCorp., Newton, Mass.); Waters (Waters Corp., Milford, Mass.).

Example 1 Wild Type Streptomyces mobaraensis Transglutaminase (MTG) GeneAcquisition and Construction of Expression Vector

A pro-gene coding for Streptomyces mobaraensis transglutaminase (MTG)was codon optimized for expression in B. megaterium based on thereported amino acid sequence (Shimonishi et al., J. Biol. Chem.,268:11565-115720 [1993]). The gene was synthesized by GenOracle andcodon-optimized using their proprietary software. The DNA was sequenceverified. The pro-MTG gene was cloned behind a B. megaterium “optimized”signal peptide plus a spacer region (6 bases encoding amino acidresidues threonine and serine into an E. coli/B. megaterium shuttlevector pSSBm, using the BsrGI/NgoMIV cloning sites. The vector pSSBm isa modified vector based on the shuttle vector pMM1525 (Boca Scientific).The signal peptide and pro-gene were under the control of an xlyosepromoter (Pxyl) regulated by the xylose repressor gene (xylR) present onthe shuttle vector. The vector contained the ‘rep U’ origin ofreplication for Bacillus and a tetracycline ampicillin resistancemarker. The vector also contained the pBR322 origin of replication andan ampicillin resistance marker for maintenance in E. coli. Theresulting plasmid (pSSBm-pre-pro-MTG) was transformed by a standardPEG-mediated method of DNA transfer into B. megaterium protoplasts. Thepre-pro-MTG sequence from the transformants was verified. Thepolynucleotide sequence of the pre-pro-MTG that includes a B. megateriumsignal peptide was cloned into the shuttle pSSBm vector and the sequenceis provided in SEQ ID NO: 6, the sequence of the pro-MTG with aC-terminus histidine purification tag comprises SEQ ID NO: 2 and thesequence of the mature MTG comprises SEQ ID NO: 4.

Example 2 B. megaterium Shake Flask Procedure

A single microbial colony of B. megaterium containing a plasmid with thepre-pro-MTG gene was inoculated into 3 ml Luria-Bertani (LB) broth (0.01g/L peptone from casein, 0.005 g/L yeast extract, 0.01 g/L sodiumchloride) containing 10 μg/mL tetracycline. Cells were grown overnightfor at least 16 hrs, at 37° C., with shaking at 250 rpm. The culture wasthen diluted into 100 mL A5 media (2 g/L (NH4)₂SO₄, 3.5 g/L KH₂HPO₄, 7.3g/L Na₂HPO₄, 1 g/L yeast extract, pH to 6.8), 100 μL of trace elementssolution (49 g/L MnCl₂.4H₂O, 45 g/L CaCl₂, 2.5 g/L (NH4)Mo₇.O₂₄.H₂O, 2.5g/L CoCl₂.6H₂O), 1.5 mL of 20% glucose, 150 μL of 1M MgSO₄, 100 μL of 10mg/mL tetracycline, 100 μL of 2.5 g/L FeSO₄.7H₂O in a 1000 ml flask toan optical density at 600 nm (OD₆₀₀) of 0.2 and allowed to grow at 37°C. Expression of the pre-pro-MTG gene was induced with 0.5% xylose(final concentration) when the OD₆₀₀ of the culture was 0.6 to 0.8 andincubated overnight, for at least 16 hrs. Cells were pelleted bycentrifugation (4000 rpm, 15 min, 4° C.). The clear media supernatantcontaining the secreted mature MTG enzyme was collected and 60 mL of thesupernatant transferred into the top cell of a Jumbosep concentrator(PES membrane, 3,000 MWCO pore size; Pall). The supernatant wascentrifuged at room temperature, 4000 rpm, until the volume became lessthan 20 mL (˜45 min). The filtrate was discarded and the remaining 20 mLof supernatant were added to the concentrate to make up a final volumeof 40 mL. The centrifugation of the 40 mL was continued at roomtemperature, 4000 rpm until the volume reached ˜20 mL (˜45 min). The 20mL of 5× concentrate were transferred into a Vivaspin 20 concentrator(PES membrane, 10,000 MWCO pore size; Vivaproducts), and centrifugeduntil the volume was ˜1 mL (˜60 min). Then, 50 mM NaOAc buffer, pH=5.0was added up to 20 mL volume (first buffer exchange), and centrifugationcontinued at room temperature, 4000 rpm until the volume was ˜1 mL (˜60min). Then, 50 mM NaOAc buffer pH=5.0 was added up to 20 mL volume(second buffer exchange), and centrifugation continued at roomtemperature, 4000 rpm until the volume was ˜5 mL (˜60 min). The 20×concentrate was mixed well and stored and stored at −20° C. MTG activitywas confirmed using the hydroxymate assay and the insulin assaysdescribed herein.

Example 3 B. megaterium High Throughput Assays to Identify Improved MTGVariants

Plasmid libraries containing variant pre-pro-MTG genes were transformedinto B. megaterium and plated on Luria-Bertani (LB) agar platescontaining 3 μg/mL tetracycline with a DM3 regeneration media (400 mMsodium succinate dibasic, pH 7.3, 0.5% casamino acids, 0.5% yeastextract, 0.4% K₂HPO₄, 0.2% KH₂PO₄, 20 mM MgCl₂, 0.5% glucose and 0.2%BSA) overlay. After incubation for at least 18 hours at 30° C., colonieswere picked using a Q-bot® robotic colony picker (Genetix) into shallow,96-well well microtiter plates containing 180 μL LB and 10 μg/mLtetracycline. Cells were grown overnight at 37° C. with shaking at 200rpm and 85% humidity. Then, 20 μL of this culture were transferred into96-well microtiter plates (deep well) containing 380 μL of subculturemedia (A5 0.3% glucose medium, as described in Example 2), with 10 μg/mLtetracycline, 1% xylose and 0.25 mM ZnSO₄. The plates were thenincubated at 37° C. with shaking at 250 rpm and 85% humidity forapproximately 15-18 hours. These plates were then centrifuged at 4000rpm for 15 minutes and the clear media supernatant containing thesecreted mature MTG enzyme was used for the high throughput hydroxymateassay.

Example 4 E. coli Expression Hosts Containing Recombinant TG Genes

The initial transglutaminase (TG) parent enzyme (SEQ ID NO: 6) of thepresent invention was codon optimized for expression in E. coli,synthesized and cloned into a pCK110900 vector (See e.g., See, U.S. Pat.No. 7,629,157 and US Pat. Appln. Publn. 2016/0244787, both of which arehereby incorporated by reference in their entireties and for allpurposes) operatively linked to the lac promoter under control of thelad repressor. The expression vector also contains the P15a origin ofreplication and a chloramphenicol resistance gene. The resultingplasmids were transformed into E. coli W3110, using standard methodsknown in the art. The transformants were isolated by subjecting thecells to chloramphenicol selection, as known in the art (See e.g., U.S.Pat. No. 8,383,346 and WO2010/144103, both of which are incorporated byreference herein, in their entirety).

Example 5 Preparation of HTP TG-Containing Wet Cell Pellets

E. coli cells containing recombinant TG-encoding genes from monoclonalcolonies were inoculated into the wells of 96 well shallow-wellmicrotiter plates containing 180μ1 LB containing 1% glucose and 30 μg/mLchloramphenicol in each well. The plates were sealed with O₂-permeableseals and cultures were grown overnight at 30° C., 200 rpm and 85%humidity. Then, 100 of each of the cell cultures were transferred intothe wells of 96-well deep-well plates containing 390 mL TB and 30 μg/mLCAM. The deep-well plates were sealed with O₂-permeable seals andincubated at 30° C., 250 rpm and 85% humidity until an OD₆₀₀ of 0.6-0.8was reached. The cell cultures were then induced by IPTG to a finalconcentration of 1 mM and incubated overnight under the same conditionsas originally used. The cells were then pelleted using centrifugation at4000 rpm for 10 min. The supernatants were discarded and the pelletsfrozen at −80° C. prior to lysis.

To lyse the cells, 225 μl lysis buffer containing 20 mM Tris-HCl buffer,pH 7.5, 1 mg/mL lysozyme, and 0.5 mg/mL PMBS was added to the cellpaste. The cells were incubated at room temperature for 2 hours withshaking on a bench top shaker. The plate was then centrifuged for 15minutes at 4000 rpm and 4° C. and the clear supernatants were used insubsequent steps.

To activate the pro-enzyme to the mature enzyme, 2 mg/mL of dispase in60 uL of 50 mM Tris-HCl buffer, pH 8.0 was added to 175 uL of above E.coli supernatant and incubated for 1 hour at 37° C.

Example 6 HTP Purification of TG Variants

HTP purification of the activated lysate was carried out in HisPur™Ni-NTA spin plate (Life Technologies, cat #88230) using manufacturer'sprotocol, with modifications, as described. First, 225 uL of dispaseactivated lysate obtained as described in Example 5 was diluted by anequal volume of binding buffer containing 50 mM Na phosphate, pH 7.5,300 mM NaCl, and 10 mM imidazole. Then, 165 uL of the diluted lysate wasapplied to HisPur™ Ni-NTA spin plate pre-equilibrated in the bindingbuffer and incubated for 10 min at room temperature followed bycentrifugation. This step was repeated once. The spin plate was thenwashed with 600 uL of washing buffer composed of 50 mM Na phosphate, pH7.5, 300 mM NaCl, and 25 mM imidazole. The purified enzyme was theneluted in 105 uL of elution buffer containing 50 mM Na phosphate, pH7.5, 300 mM NaCl, and 250 mM imidazole.

Example 7 Preparation of Lyophilized Lysates from Shake Flask (SF)Cultures

Selected HTP cultures grown as described above were plated onto LB agarplates with 1% glucose and 30 μg/ml CAM, and grown overnight at 37° C. Asingle colony from each culture was transferred to 6 ml of LB with 1%glucose and 30 μg/ml CAM. The cultures were grown for 18 h at 30° C.,250 rpm, and subcultured approximately 1:50 into 250 ml of TB containing30 μg/ml CAM, to a final OD₆₀₀ of 0.05. The cultures were grown forapproximately 195 minutes at 30° C., 250 rpm, to an OD₆₀₀ between0.6-0.8 and induced with 1 mM IPTG. The cultures were then grown for 20h at 30° C., 250 rpm. The cultures were centrifuged 4000 rpm×20 min. Thesupernatant was discarded, and the pellets were resuspended in 30 ml of20 mM Tris-HCl, pH 7.5. The cells were pelleted (4000 rpm×20 min) andfrozen at −80° C. for 120 minutes. Frozen pellets were resuspended in 30ml of 20 mM TRIS-HCl pH 7.5, and lysed using a Microfluidizer® processorsystem (Microfluidics) at 18,000 psi. The lysates were pelleted (10,000rpm×60 min) and the supernatants were frozen and lyophilized to generateshake flake (SF) enzymes.

Example 8 Improvements in Activity of Transglutaminase Expressed by B.megaterium

HTP B. megaterium cell pellets obtained as described in Example 3 werecentrifuged for 15 minutes at 4000 rpm and 4° C. and the clear mediasupernatants were used in subsequent biocatalytic reactions. HTPreactions were carried out in 96 well deep well plates containing 100 μLof 0.2 M Tris-HCl, pH 8.0, 0.04 M glutamyl donor substrate Z-Gln-Gly(Sigma, C6154), 0.1 M hydroxylamine, 0.01 M glutathione, and 5 μl HTP B.megaterium culture lysate supernatant. The HTP plates were incubated ina Thermotron® titre-plate shaker (3 mm throw, model #AJ185, Infors) at37° C., 100 rpm, for 35 min. The reactions were quenched with 100 μl 0.8M HCl containing 0.3 M trichloraacetic acid and 2 M FeCl₃.6H₂O.Absorbance of the samples was recorded at 525 nm.

The fold improvement over positive control (FIOPC) was calculated as theabsorbance of the product normalized by the absorbance of thecorresponding backbone under the specified reaction conditions. Theresults are shown in Table 8.1, below.

TABLE 8.1 Transglutaminase HTP Activity Results SEQ ID NO: Amino AcidDifferences Activity Improvement (nt/aa) (Relative to SEQ ID NO: 6)(FIOP)^(†) on Glutathione 7/8 G327R ++  9/10 Y101G/Q201K/R212K/S287G ++11/12 Y101G/S287G ++ 13/14 S79K ++ 15/16 Y101G/Q201K/R285Q ++ 17/18Y101G ++ ^(†)Levels of increased activity or selectivity were determinedrelative to the reference polypeptide of SEQ ID NO: 6, and defined asfollows: “+” > than 1.2-fold but less than 1.5-fold increase; “++” >than 1.5-fold but less than 2-fold; “+++” > than 2-fold.

Example 9 Improvements in Activity Trasglutaminase Expressed in E. coli

Libraries of the parent enzyme (SEQ ID NO: 2) containing engineeredgenes were produced using well established techniques known in the art(e.g., saturation mutagenesis and recombination of previously identifiedbeneficial mutations). The polypeptides encoded by each gene wereproduced in HTP as described in Example 4 and the soluble lysate wasgenerated as described in Example 5. The following assays were used toevaluate the activity of these variant polypeptides.

Assay A: Glutathione Assay

HTP reactions were carried out in 96-well deep-well plates containing100 μL of 0.2 M Tris-HCl, pH 8.0, 0.04 M Z-Gln-Gly, 0.1 M hydroxylamine,0.01 M glutathione, and 10 uL of activated lysate supernatant. The HTPplates were incubated in a Thermotron® titre-plate shaker (3 mm throw,model #AJ185, Infors) at 37° C., 300 rpm, for 30 min. The reactions werequenched with 100 μl of quenching solution containing 0.8 M HCl, 0.3 Mtrichloroacetic acetate, and 2 M FeCl₃.6H₂O, mixed for 10 minutes usinga bench top shaker. The plates were then centrifuged at 4000 rpm for 5minutes and absorbance at 525 nm recorded. The fold improvement overpositive control (FIOPC) was calculated as the UV signal of the variantsnormalized by that of the corresponding backbone under the specifiedreaction conditions.

Assay B: Insulin Assay

HTP reactions were carried out in 96-well deep-well plates containing200 μL of 0.1 M Tris-HCl, pH 8.0, 1 g/L insulin, 25 mM EDTA, 1.25 mMZ-Gln-donor substrate, and 70 uL of purified lysate as described inExample 6. The HTP plates were incubated in a Thermotron® titre-plateshaker (3 mm throw, model #AJ185, Infors) at 30° C., 300 rpm, for 24hours. The reactions were quenched with 200 μl DMSO and mixed for 5minutes using a bench top shaker. The plates were then centrifuged at4000 rpm for 5 minutes, and the supernatants loaded into LC-MS foranalysis. The LC-MS and UV signals were both collected. The foldimprovement over positive control (FIOPC) was calculated as the UVsignal of the modified insulin in variants normalized by that of thecorresponding backbone under the specified reaction conditions.

TABLE 9.1 Assay Results for Transglutaminase Variants Activity ActivityImprovement Improvement SEQ ID (FIOP)^(†) on (FIOP)^(‡) on NO: AminoAcid Differences Glutatione Insulin (nt/aa) (Relative to SEQ ID NO: 2)(Assay A) (Assay B) 19/20 S48K/G203L/G296L/N343R/ ++++ E346H/K373M 21/22S48K/Q170K/G203L/E346H/ ++ ++++ K373M 23/24 S48K/Q170K/G203L/R254Q/ ++++++ E346H 25/26 S48K/G203L/N343R/E346H/ + ++++ K373M 27/28S48K/G203L/R254Q/E346H/ +++ ++++ K373M 29/30 S48K/Q170K/G203L/N343R/++++ E346H 31/32 S48K/Q170K/G203L/G296L/ ++++ N343R/E346H 33/34S48K/G203L/N343R/E346H ++++ 35/36 N343R/E346H/K373M ++++ 37/38S48K/Q170K/G203L/G296L/ +++ ++++ E346H/K373M 39/40S48K/G203L/G296L/E346H/ +++ ++++ K373M 41/42 S48K/Q170K/G203L/R254Q/ +++++++ G296L/E346H/K373M 43/44 S48K/G203L/E346H ++ ++++ 45/46S48K/Q170K/G203L/R254Q/ ++++ E346H/K373M 47/48 S48K/G203L/E346H/K373M++++ 49/50 S48K/Q170K/G203L/R254Q/ ++ ++++ G296L/E346H 51/52G203L/N343R/E346H ++++ 53/54 S48K/Q170K/G296L/N343R/ ++++ E346H 55/56S48K/G203L/R254Q/E346H ++ ++++ 57/58 S48K/G203L/R254Q/G296L/ +++ ++++E346H/K373M 59/60 S48K/Q170K/G203L/E346H ++ ++++ 61/62S48K/G203L/G296L/E346H ++++ 63/64 S48K/N343R/E346H ++++ 65/66S48K/R254Q/E346H ++++ 67/68 S48V/R67E/G203V/S256G/ +++ +++ G296R/K373V69/70 S48K/Q170K/N343R/E346H +++ 71/72 Q170K/G203L/N343R/E346H +++ 73/74Q170K/G203L/R254Q/G296L/ ++ +++ N343R/E346H 75/76 G203L/E346H +++ 77/78S48V/R67E/Y70G/S256G/ +++ +++ G296R/S345E/K373V 79/80S48K/G203L/R254Q/G296L/ +++ +++ N343R/K373M 81/82 F297W/E346A +++ 83/84S48V/S256G/G296R ++ +++ 85/86 P68A/R282K/F297W/E346A +++ 87/88F136Y/P215N/H234Y/F297W/ +++ E346A 89/90 P68A/E74T/S190G/P215N/ +++E346A 91/92 E74T/F136Y/E346A +++ 93/94 P215N/H234Y/F297W/E346A +++ 95/96G203L/N343R ++ ++ 97/98 S48V/R67E/Y70G/G203V/ +++ ++ S256G/G296R/S345E 99/100 F136Y/S190G/P215N/F297W/ ++ E346A 101/102 E74T/E346A ++ 103/104Q170K/G203L/R254Q/N343R/ ++ ++ K373M 105/106 S48V/G296R/K373V ++ ++107/108 P68A/F297W/E346A ++ 109/110 P68A/V158I/E174D/H234Y/ ++R282K/F297W/E346A 111/112 E174D/R282K/F297W/E346A ++ 113/114E74T/F136Y/E174D/F297W/ ++ E346A 115/116 E74T/S255R/E346A ++ 117/118E174D/P215N/H234Y/F297W/ ++ E346A 119/120 S48V/R67E/Y70G/R181K/ +++ ++G296R/S345E/K373V 121/122 P215H/S255R/F297W/E346A ++ 123/124P215N/S255R/F297W/E346A ++ 125/126 S48K/G203L/G296L/N343R/ +++ ++ K373M127/128 S48V/R181K/G296R + ++ 129/130 P68A/F136Y/P215N/F297W/ ++ E346A131/132 P215N/E346A ++ 133/134 S190G/F297W/E346A ++ 135/136F136Y/F297W/E346A ++ 137/138 E174D/S190G/H234Y/F297W/ ++ E346A 139/140S48V/R67E/Y70G/R181K/ +++ ++ G203V/S256G 141/142 E74T/F136Y/E174D/R282K/++ E346A 143/144 S255R/F297W/E346A ++ 145/146 F136Y/E174D/P215N/S255R/++ R282K/F297W/E346A 147/148 V158I/P215N/S255R/E346A ++ ++ 149/150P215N/F297W/E346A ++ 151/152 P68A/V158I/P215N/F297W/ ++ E346A 153/154F136Y/V158I/P215N/F297W/ ++ E346A 155/156 H234Y/S255R/E346A ++ 157/158S255R/E346A ++ 159/160 E346A ++ 161/162 F136Y/V158I/S190G/P215N/ ++S255R/F297W/E346A 163/164 F136Y/P215N/F297W ++ 165/166P68A/F136Y/P215N/S255R/ ++ R282K/F297W/E346A 167/168P68A/P215N/F297W/E346A ++ 169/170 S48K/Y70L/G203L/R254Q/ +++ ++G296L/N343R 171/172 S48V/G203V/S256G ++ ++ 173/174S190G/S255R/R282K/E346A ++ 175/176 S48K/G203L/R254Q/G296L +++ ++ 177/178S48V/R67E/G203V/S256G/ ++ ++ S345E 179/180 V158I/P215N/E346A ++ 181/182S48V/G203V/S256G/G296R/ ++ S345E 183/184 S48V/Y70N/G203V/K373V ++ ++185/186 S48V/Y70G/G203V/S256G/ +++ ++ S345E/K373V 187/188 S48V/R67E/Y70G++ ++ 189/190 S48K/G203L ++ ++ 191/192 E174D/P215N/S255R/F297W/ ++ E346A193/194 S48V/R181K/S256G/G296R/ ++ S345E 195/196S48V/R67E/Y70G/G203V/S345E ++ ++ 197/198 S48V/R67E/Y70G/R181K/ ++ ++S256G/S345E 199/200 S48V/R67E/Y70N/S256G ++ ++ 201/202S48K/Q170K/G203L/K373M ++ ++ 203/204 S48V/G203V ++ 205/206S48V/R181K/G296R/S345E ++ 207/208 R67E/G296R/S345E ++ ++ 209/210 S48V ++211/212 S48V/S256G/G296R/S345E ++ 213/214 S48V/R67E/Y70N/G203V/ +++ ++S256G/S345E/G354H/K373L 215/216 S48K/Q170K/G203L + ++ 217/218F136Y/P215N/H234Y/R282K/ ++ F297W 219/220 S48V/R181K ++ 221/222S48K/R254Q/G296L ++ ++ 223/224 R67E/S256G + ++ 225/226S48K/Q170K/G296L + + 227/228 E74T/V158I/S255R/F297W + 229/230S48V/G296R/S345E + 231/232 S48V/S256G + 233/234 P68A/H234Y ++ + 235/236S48V/R181K/G203V/S256G/ ++ + S345E 237/238 S48K/Q170K/R254Q + 239/240S48K/Y70D/Q170K/G203L ++ + 241/242 S48V/G203V/S345E + 243/244G203L/G296L ++ + 245/246 P68A/F136Y/H234Y + 247/248 S48V/S345E/K373L +249/250 S48V/Y70N/G203V/S256G/S345E ++ + 251/252 P215N/F297W + 253/254S48V/R181K/G203V/S345E + ^(†)Levels of increased activity or selectivitywere determined relative to the reference polypeptide of SEQ ID NO: 2.and defined as follows: “+” > than 1.2-fold but less than 1.5-foldincrease: “++” > than 1.5-fold but less than 2-fold; “+++” > than2-fold. ^(‡)Levels of increased activity or selectivity were determinedrelative to the reference polypeptide of SEQ ID NO: 2, and defined asfollows: “+” > than 1.2-fold but less than 2.0-fold increase: “++” >than 2.0-fold but less than 5-fold: “+++” > than 5-fold but less than10-fold; “++++” > than 10-fold.

TABLE 9.2 Transglutaminase Variant Activity Results Activity ActivityImprovement Improvement SEQ ID (FTOP)^(†) on (FIOP)^(‡) on NO: AminoAcid Differences Glutathione Insulin (nt/aa) (Relative to SEQ ID NO: 2)(Assay A) (Assay B) 493/494 S48K/G203L/R254Q/N343R + ++ 495/496S48V/R67E/G203V/S256G/ ++ ++ G296R/K373V/G378D 497/498S48V/R67E/G203V/G296R/ ++ ++ K373V 499/500 S48V/Y70G/G203V/G296R/ ++ ++K373V 501/502 S48V/Y70G/G203V/S256G/ ++ ++ G296R/K373V 503/504S48V/G203V/S256G/G296R/ ++ ++ K373V 505/506 S48V/R67E/S256G/G296R/ ++ ++K373V 507/508 S48V/G203V/G296R/K373V/ + ++ Q374L 509/510S48V/R67E/Y70G/G203V/ ++ ++ S256G/G296R/K373V 511/512S48V/G203V/G296R/K373V ++ ++ 513/514 S48V/R67E/Y70G/R181K/ ++ ++G203V/S256G/G296R/K373V 515/516 S48V/R67E/R181K/G203V/ ++ ++S256G/G296R/K373V 517/518 S48V/Y70G/S256G/G296R/ ++ ++ K373V 519/520S48V/Y70G/R181K/G203V/ ++ ++ S256G/G296R/K373V 521/522S48V/G203V/S256G/G296R ++ ++ 523/524 A36E/S48K/G203L/R254Q/ ++ ++ E346H525/526 S48V/Y70G/G203V/G296R ++ ++ 527/528 S48V/R181K/G203V/G296R + ++529/530 S48V/S256G/G296R/K373V + ++ 531/532 S48V/R181K/S256G/G296R/ + ++K373V 533/534 S48V/Y70G/R181K/G203V/ ++ ++ G296R/K373V 535/536S48V/R181K/G203V/S256G/ ++ ++ K373V 537/538 G203V/S256G/G296R/K373V ++++ 539/540 S48V/R67E/R181K/S256G/ + ++ G296R 541/542G203L/R254Q/N343R/E346H/ + ++ K373M 543/544 Y70G/G203V/S256G/G296R/ ++++ K373V 545/546 R67E/R181K/G203V/S256G/ ++ ++ G296R/K373V 547/548S48V/Y70G/G296R/K373V + ++ 549/550 R67E/Y70G/R181K/G203V/ ++ ++S256G/G296R/K373V 551/552 Y70G/R181K/G203V/G296R/ ++ ++ K373V 553/554S48V/Y70G/G203V/K373V ++ ++ 555/556 S48V/R67E/R181K/G203V/ ++ ++S256G/K373V 557/558 S48V/R67E/G203V/S256G/ ++ ++ K373V 559/560S48V/Y70G/G203V/S256G/ ++ ++ K373V 561/562 R181K/G203V/S256G/G296R/ ++++ K373V 563/564 R67E/G203V/S256G/G296R/ ++ ++ K373V 565/566G203V/G296R/K373V + ++ 567/568 R67E/S256G/G296R/K373V + ++ 569/570S48V/S256G/K373V + ++ 571/572 R181K/G203V/G296R/K373V + ++ 573/574S48V/G203V/K373V + ++ 575/576 A33D/R67E/Y70G/R181K/ ++ ++G203V/S256G/G296R/K373V 577/578 S48V/R181K/G203V/K373V + ++ 579/580S48V/G203V/S256G/K373V ++ ++ 581/582 G203L/R254Q/E346H/K373M + ++583/584 R67E/R181K/G203V/S256G/ ++ ++ G296R 585/586G203V/S256G/G296R/K373V/ + + H386Y 587/588 G203L/R254Q/E346H ++ +589/590 S256G/G296R + + 591/592 Y70G/R181K/G203V/S256G/ ++ + G296R/K373V593/594 R67E/T70G/R181K/S256G/ + + G296R/K373V 595/596G203V/S256G/G296R + + 597/598 Y70G/G203V/G296R/K373V + + 599/600R181K/S256G/G296R/K373V + + 601/602 S48V/Y70G/R181K/G203V/ ++ +S256G/K373V 603/604 G203L/P224T/R254Q/K373M + + 605/606G203V/S256G/K373V + + 607/608 S256G/G296R/K373V + + 609/610S48K/G203L/R254Q/N343R/ + + E346H/K373M 611/612R181K/G203V/S256G/G296R + + 613/614 S48K/R254Q/N343R/E346H/ + + K373M615/616 R67E/R181K/G203V/S256G/ + + K373V 617/618 S48V/K373V + 619/620S256G/K373V + + 621/622 R254Q/E346H/K373M + + 623/624S48K/G203L/R254Q/N343R/ ++ + K373M 625/626 S48K/G203L/N343R/K373M + +627/628 R181K/G203V/S256G/K373V + + 629/630 G203V/N209Y/S256G/K373V + +631/632 R181K/G203V/K373V + + 633/634 G203L/E346H/K373M + + 635/636G203V/S256G + + 637/638 H234Y/R282K/E346A + + 639/640Y70G/G203V/S256G/K373V ++ + 641/642 R181K/G203V/S256G + + 643/644F136Y/P215N/R282K/E346A + + 645/646 G203V/K373V + + 647/648R67E/Y70G/R181K/K373V + 649/650 R254Q/E346H + 651/652 Y70G/G203V +653/654 S48K/G203L/R254Q/N343R/ + A355T/K373M 655/656S48K/R254Q/E346H/K373M + 657/658 G203V/S256G/G296R/H320Y/ + K373V659/660 G203L/R254Q/N343R/K373M ++ 661/662 S48K/R254Q/N343R/K373M +663/664 S48K/G203L/R254Q/E346D/ + K373M 665/666 G203L/K373M + 667/668S48K/R254Q + 669/670 K373M + 671/672 G203L/R254Q/K373M ++ 673/674S48K/G203L/R254Q ++ 675/676 S48K/G203L/K373M ++ 677/678S48K/R254Q/K373M + 679/680 R254Q/K373M + 681/682 G203L/R254Q + 683/684S48K/G203L/R254Q/K373M ++ 685/686 R254Q + 687/688E74T/F136Y/H234Y/R282K/ ++ F297W/E346A 689/690 E74T/F136Y/P215N/H234Y/++ R282K/F297W/E346A 691/692 E74T/P215N/H234Y/R282K/ ++ F297W/E346A693/694 E74T/F136Y/P215N/R282K/ ++ F297W/E346A 695/696P215N/H234Y/R282K/F297W/ ++ E346A 697/698 E74T/F136Y/P215N/H234Y/ ++R282K/E346A 699/700 E74T/F136Y/P215N/H234Y/ ++ F297W/E346A 701/702E74T/F136Y/R282K/F297W/ ++ E346A 703/704 E74T/F136Y/P215N/F297W/ ++E346A 705/706 E74T/P215N/R282K/F297W/ ++ E346A 707/708E74T/F136Y/P215N/H234Y/ + F297W/N343Y/E346A 709/710 N343R/K373M +711/712 E74T/F136Y/P215N/H234Y/ + E346A 713/714S48V/R181K/G203V/S256G/ + G296R/K373V 715/716 F136Y/P215N/H234Y/R282K/ +F297W/E346A 717/718 F136Y/H234Y/E346A + 719/720 E74T/P215N/E346A +721/722 E74T/F136Y/P215N/E346A + 723/724 F136Y/H234Y/F297W/E346A +725/726 F136Y/P215N/R282K/F297W/ + E346A 727/728 F136Y/P215N/E346A +729/730 P215N/H234Y/R282K/E346A + 731/732 F136Y/P215N/F297W/E346A +733/734 E74T/F136Y/H234Y/E346A + 735/736 R282K/F297W/E346A + 737/738P215N/H234Y/E346A + 739/740 E74T/F136Y/P215N/R282K/ + E346A 741/742E74T/F136Y/P215N/H234Y/ + F297W 743/744 K373V + 745/746 R181K/G296R +747/748 S48K/A176T/G203L/R254Q/ + E346H/K373M 749/750F136Y/P215N/R282K/F297W + 751/752 F136Y/H234Y/F297W + 753/754E74T/P215N + 755/756 F136Y/R282K/F297W + ^(‡)Levels of increasedactivity or selectivity were determined relative to the referencepolypeptide of SEQ ID NO: 2. and defined as follows: “+” > than 1.2-foldbut less than 2.0-fold increase; “++” > than 2.0-fold but less than5-fold; “+++” > than 5-fold but less than 10-

Example 10 Activity Improvement in Transglutaminase Variants Expressedin E. coli

Libraries of the parent enzyme (SEQ ID NO: 34) containing engineeredgenes were produced using well established techniques known in the art(e.g., saturation mutagenesis and recombination of previously identifiedbeneficial mutations). The polypeptides encoded by each gene wereproduced in HTP as described in Example 4, and the soluble lysate wasgenerated as described in Example 5.

HTP reactions were carried out in 96-well deep-well plates containing200 μL of 0.1 M Tris-HCl, pH 8.0, 1 g/L insulin, 25 mM EDTA, 5 mM lysinedonor substrate, and 70 uL of purified lysate as described in Example 6.The HTP plates were incubated in a Thermotron® titre-plate shaker (3 mmthrow, model #AJ185, Infors) at 30° C., 300 rpm, for 22 hours. Thereactions were quenched with 200 μl DMSO and mixed for 5 minutes using abench top shaker. The plates were then centrifuged at 4000 rpm for 5minutes, supernatant loaded into LC-MS for analysis. The foldimprovement over positive control (FIOPC) was calculated as the mass ofinsulin modified with one lysine donor in variants normalized by that ofthe corresponding backbone under the specified reaction conditions.

TABLE 10.1 Transglutaminase Variant Activity SEQ ID NO: Amino AcidDifferences Activity Improvement (nt/aa) (Relative to SEQ ID NO: 34)(FIOP)^(‡) on Insulin 255/256 D50R ++ 257/258 D50A ++ 259/260 L331H ++261/262 L331P + 263/264 D50Q ++ 265/266 K48S/D49W ++ 267/268 L331V +269/270 D50F + 271/272 S292R + 273/274 T291C + 275/276 S330Y + 277/278L331R + 279/280 S330H + 281/282 D49Y + ^(‡)Levels of increased activityor selectivity were determined relative to the reference polypeptide ofSEQ ID NO: 34, and defined as follows: “+” > than 1.2-fold but less than2.0-fold increase; “++” > than 2.0-fold but less than 5-fold; “+++” >than 5-fold but less than 10-fold, “++++” > than 10-fold.

Example 11 Activity Improvement in Transglutaminase Variants Expressedin E. coli

Libraries of the parent enzyme (SEQ ID NO: 256) containing engineeredgenes were produced using well established techniques known in the art(e.g., saturation mutagenesis and recombination of previously identifiedbeneficial mutations). The polypeptides encoded by each gene wereproduced in HTP as described in Example 4, and the soluble lysates weregenerated as described in Example 5.

HTP reactions were carried out in 96-well deep-well plates containing200 μL of 0.1 M Tris-HCl, pH 8.0, 2 g/L insulin, 25 mM EDTA, 5 mM lysinedonor substrate, 10% acetonitrile, and 70 uL of purified lysate producedas described in Example 6. The HTP plates were incubated in aThermotron® titre-plate shaker (3 mm throw, model #AJ185, Infors) at 30°C., 300 rpm, for 22 hours. The reactions were quenched with 200 μl DMSOand mixed for 5 minutes using a bench top shaker. The plates were thencentrifuged at 4000 rpm for 5 minutes, and supernatants loaded intoLC-MS for analysis. The fold improvement over positive control (FIOPC)was calculated as the mass of insulin modified with one lysine donor invariants normalized by that of the corresponding backbone under thespecified reaction conditions.

TABLE 11.1 Transglutaminase Variant Assay Results Activity SEQ IDImprovement NO: Amino Acid Differences (FIOP)^(‡) on (nt/aa) (Relativeto SEQ ID NO: 256) Insulin 283/284 K48S/D49W/R50A/L331V +++ 285/286K48S/D49Y/R50A/T291C/S292R/L331V +++ 287/288K48V/L203V/H234Y/H346A/K373M +++ 289/290 K48S/D49W/R50A/S292R ++ 291/292K48V/L203V/H234Y/S256G/H346A/K373M ++ 293/294 L203V/K373M ++ 295/296K48S/D49W/S330Y/L331V ++ 297/298 R67E/Y70G/E74T/P215H/H234Y/F297W/ ++H346A/K373L 299/300 K48S/D49W/R50A/S349R ++ 301/302 R67E/F297W/H346A ++303/304 R67E/E74T/P215H/H346A/K373V ++ 305/306R67E/Y70G/E74T/F136Y/L203V/P215H/ ++ S256G/H346A/K373M 307/308R67E/P215H/H234Y/F297W/H346A/K373V ++ 309/310K48V/R67E/E74T/H234Y/F297W/H346A/ ++ K373M 311/312 S292R/S330Y/L331P ++313/314 R67E/Y70G/E74T/P215H/S256G/K373M ++ 315/316K48S/D49G/R50A/S292R/L331P ++ 317/318 K48V/R67E/H346A/K373M ++ 319/320R67E/E74T/P215H/S256G/F297W/H346A/ ++ K373L 321/322R67E/Y70G/F136Y/L203V/F297W/H346A/ ++ K373M 323/324R67E/Y70G/L203V/P215H/S256G/H346A/ ++ K373L 325/326K48V/R67E/Y70G/H234Y/S256G/R282K/ ++ F297W/H346A 327/328K48V/R67E/E74T/H346A ++ 329/330 N27S/K48V/R67E/Y70G/H346A/K373L ++331/332 D49W/R50A/L331V ++ 333/334 R67E/Y70G/E74T/L203V/P215H/H234Y/ ++H346A/K373V 335/336 N27S/K48V/R67E/Y70G/F136Y/L203V/ ++P215H/S256G/R282K/H346A/K373V 337/338 R67E/F136Y/L203V/P215H/S256G/ ++H346A/K373V 339/340 Y70G/E74T/L203V/P215H/H346A/K373V ++ 341/342R67E/Y70G/P215H ++ 343/344 K48V/R67E/Y70G/L203V/P215H/H234Y/ ++S256G/H346A 345/346 K48V/R67E/L203V/H346A/K373M ++ 347/348N27S/K48V/R67E/E74T/L203V/S256G/ ++ H346A/K373M 349/350 R67E/E74T/F136Y++ 351/352 N27S/R67E/H234Y/G296R/K373M ++ 353/354K48V/R67E/P215H/R282K/F297W/ ++ H346A/K373M 355/356 F136Y/H346A/K373M ++357/358 R67E/F136Y/L203V/S256G/H346A/ ++ K373M 359/360 F297W/K373M ++361/362 K48V/E74T/H234Y/S256G/F297W/ ++ H346A/K373V 363/364K48V/Y70G/E74T/F297W/H346A/ ++ K373M 365/366K48V/Y70G/P215H/H234Y/S256G/ ++ H346A/K373M 367/368N27S/K48V/R67E/Y70G/E74T/ ++ H234Y/S256G/R282K/H346A/K373L 369/370K48V/R67E/E74T/L203V/H234Y/ ++ S256G/R282K/H346A/K373M 371/372R67E/Y70G/L203V/K373M ++ 373/374 R67E/L203V/F297W/H346A/K373M ++ 375/376R67E/E74T/S256G/H346A/K373M ++ 377/378 H234Y/H346A/K373M ++ 379/380K48V/Y70G/L203V/P215H/S256G/ ++ R282K/H346A/K373V 381/382K48V/F136Y/S256G/H346A/K373M ++ 383/384 K48V/S256G/K373L ++ 385/386K48V/P215H/H346A/K373M ++ 387/388 K48V/P215H/H234Y/H346A/K373V ++389/390 K48V/R67E/Y70G/H346A ++ 391/392 K48S/D49Y/R50Q/S292R/L331V ++393/394 S292R/S330Y/L331V ++ 395/396 E295R/F297Y/A333P ++ 397/398D49W/R50A/L331V/S349R ++ 399/400 K48V/I203V/H234Y/S256G/F297W/ ++H346A/K373V 401/402 D49G/R50A/S292R/L331V ++ 403/404K48V/E74T/L203V/H234Y/S256G/ ++ H346A/K373V 405/406 S292R/S349R ++407/408 K48V/H234Y/S256G/G296R/H346A/ ++ K373M 409/410 L203V/H234Y/H346A++ 411/412 D49G/R50Q/S292R/L331V/S349R ++ 413/414 S292R/L331V/S349R ++415/416 S292R ++ 417/418 K48V/L203V/G296R/K373M ++ 419/420 S330Y/L331P++ 421/422 K48V/R67E/H234Y/S256G/F297W/ ++ H346A/K373V 423/424K48A/P287S/S292K/F297Y ++ 425/426 K48V/H234Y/S256G/H346A/K373M ++427/428 K48V/R67E/H234Y/S256G/H346A/ ++ K373M 429/430R67E/E74T/L203V/H234Y/S256G ++ 431/432 L203V/H234Y/H346A/K373V ++433/434 R67E/L203V/H234Y/S256G/H346A/ ++ K373V 435/436 R50A + 437/438A45S/S292K/N328E + 439/440 S292R/L331V + 441/442 N328E/A333P + 443/444L331V/S349R + 445/446 A333P + 447/448 L331V + 449/450K48A/P287S/F297Y/N328E/A333P + 451/452 K373V + 453/454 H346A/K373V +455/456 K48A + 457/458 K48A/S292K + 459/460 P287S + 461/462K48A/R284G/S292K/A333P + 463/464 A45S/P287S/N328E/A333P + 465/466P287S/E295R/F297Y + 467/468 P287S/S292K/F297Y + 469/470 S292K + 471/472F136Y + 473/474 E295R + 475/476 K48A/S292K/F297Y + 477/478 S292K/F297Y +479/480 F297Y/N328E + 481/482 P287S/S292K/E295R/F297Y + 483/484P287S/S330G/A333P + 485/486 P287S/S292K + 487/488 K373M + 489/490H234Y/R282K + 491/492 S330Y + ^(‡)Levels of increased activity orselectivity were determined relative to the reference polypeptide of SEQID NO: 256, and defined as follows: “+” > than 1.2-fold but less than2.0-fold increase; “++” > than 2.0-fold but less than 5-fold, “+++” >than 5-fold but less than 10-fold: “++++” > than 10-fold.

Example 12 Analytical Detection of TG Production Formation

Data described in Examples 8-11 were collected using analytical methodsin Tables 12.1 or 12.2. LC-MS analysis methods for the product resultingin the modification of insulin by the glutamine Z-donor are provided inTable 12.1. The HTP assay mixtures prepared and formation of themodified insulin product compound detected by LC-MS-UV using theinstrumental parameters and conditions shown in Table 12.1. The mass ofthe product was used to determine the substrate and product peaks andthe UV signal was used to quantify each species and compare to thepositive control and calculate FIOP.

TABLE 12.1 Analytical Method Instrument Thermo LXQ Column WatersX-bridge C18 column: 50 × 3.0 mm, 5 um, with Phenomenex C18 guardCartridge: 5 × 3.0 mm, 5 μm Mobile Gradient (A: 0.2% formic acid inwater; B: 0.2% Phase formic acid in MeCN) Time(min) % A 0.0 75 1.0 754.0 70 5.0 5.0 6.0 75 7.0 75 Flow Rate 0.7 mL/min Run Time 7 min Column45° C. Temperature Injection 10 μL Volume MS LXQ; divert flow from MSbetween 0-0.5 min. Detection BP extracted ions for: insulin product (+6,+5, +4 species) = 969.2, 1163.0, 1453.0 modified insulin product (+5. +4species) = 1227.0, 1533.0 MS MS Polarity: Positive; Ionization: ESI;Mode: Q1 Scan Conditions from 200-2000; Source voltage: 5; Sheath gas:60; Aux gas: 15; Cap temp: 350; Cap V: 35; Tube lens: 110. UV UV 280 nmDetection Detector: PDA (Thermo LXQ); Wavelength Step = 1 nm; Filterrise time = 1 sec; Sample rate = 5 Hz; Filter bandwidth = 1 nm RetentionInsulin product at 3.1 min; modified insulin product time at 4.6 min

LC-MS Analysis for the product resulting in the modification of insulinby the lysine donor substrate is shown in Table 12.2. The HTP assaymixtures prepared and formation of the modified insulin product compounddetected by LC-MS-UV using the instrumental parameters and conditionsare provided in Table 12.2. The mass of the product was used todetermine the substrate and product peaks and the UV signal was used toquantify each species and compare to the positive control and calculateFIOP.

TABLE 12.2 Analytical Method Instrument Thermo LXQ Column WatersX-bridge C18 column: 50 × 3.0 mm, 5 um, with Phenomenex C18 guardCartridge: 5 × 3.0 mm, 5 μm Mobile Gradient (A: 0.2% formic acid inwater; B: 0.2% Phase formic acid in MeCN) Time(min) % A 0.0 75 1.0 754.0 70 5.0 5.0 6.0 75 7.0 75 Flow Rate 0.7 mL/min Run Time 7 min Column45° C. Temperature Injection 10 μL Volume MS LXQ; divert flow from MSbetween 0-0.5 min. Detection BP extracted ions for: insulin product (+6,+5, +4 species) = 969.3, 1162.8, 1453.0 mono-lysine modified insulinproduct (+6, +5, +4 species) = 990.8, 1188.6, 1485.3 di-lysine modifiedinsulin product (+6, +5, +4 species) = 1012.3, 1214.4, 1517.5 tri-lysinemodified insulin product (+6, +5, +4 species)= 1033.7, 1239.9, 1549.5 MSMS Polarity: Positive; Ionization: ESI; Mode: QI Scan Conditions from200-2000; Source voltage: 5; Sheath gas: 60; Aux gas: 15; Cap temp: 350;Cap V: 35; Tube lens: 110 Retention Insulin product at 3.0 min;mono-lysine modified time insulin product at 2.7 mm; di-lysine modifiedinsulin product at 2.5 min; tri-lysine modified insulin at 2.4 min

While the invention has been described with reference to the specificembodiments, various changes can be made and equivalents can besubstituted to adapt to a particular situation, material, composition ofmatter, process, process step or steps, thereby achieving benefits ofthe invention without departing from the scope of what is claimed.

For all purposes in the United States of America, each and everypublication and patent document cited in this disclosure is incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an indication that any such document is pertinent prior art, nor doesit constitute an admission as to its contents or date.

We claim:
 1. An engineered polynucleotide encoding an engineeredtransglutaminase, wherein said engineered transglutaminase comprises apolypeptide sequence comprising at least 85% or more sequence identityto SEQ ID NO: 2, 6, 34, and/or
 256. 2. The engineered polynucleotide ofclaim 1, wherein said engineered polynucleotide encodes an engineeredtransglutaminase comprising a polypeptide sequence having at least 85%or more sequence identity to SEQ ID NO:6, and at least one substitutionor substitution set at one or more positions selected from positions 79,101, 101/201/212/287, 101/201/285, 101/287, and 327, wherein saidpositions are numbered with reference to SEQ ID NO:6.
 3. The engineeredpolynucleotide of claim 1, wherein said engineered polynucleotideencodes an engineered transglutaminase comprising a polypeptide sequencehaving at least 85% or more sequence identity to SEQ ID NO:2, and atleast one substitution or substitution set at one or more positionsselected from positions 48, 48/67/70, 48/67/70/181/203/256,48/67/70/181/256/345, 48/67/70/181/296/345/373,48/67/70/203/256/296/345, 48/67/70/203/256/345/354/373,48/67/70/203/345, 48/67/70/256, 48/67/70/256/296/345/373,48/67/203/256/296/373, 48/67/203/256/345, 48/70/170/203,48/70/203/254/296/343, 48/70/203/256/345/373, 48/70/203/256/345,48/70/203/373, 48/170/203, 48/170/203/254/296/346,48/170/203/254/296/346/373, 48/170/203/254/346/373, 48/170/203/254/346,48/170/203/296/343/346, 48/170/203/296/346/373, 48/170/203/343/346,48/170/203/346, 48/170/203/346/373, 48/170/203/373, 48/170/254,48/170/296, 48/170/296/343/346, 48/170/343/346, 48/181,48/181/203/256/345, 48/181/203/345, 48/181/256/296/345, 48/181/296,48/181/296/345, 48/203, 48/203/254/296, 48/203/254/296/343/373,48/203/254/296/346/373, 48/203/254/346, 48/203/254/346/373, 48/203/256,48/203/256/296/345, 48/203/296/343/346/373, 48/203/296/343/373,48/203/296/346, 48/203/296/346/373, 48/203/343/346, 48/203/343/346/373,48/203/345, 48/203/346, 48/203/346/373, 48/254/296, 48/254/346, 48/256,48/256/296, 48/256/296/345, 48/296/345, 48/296/373, 48/343/346,48/345/373, 67/256, 67/296/345, 68/74/190/215/346,68/136/215/255/282/297/346, 68/136/215/297/346, 68/136/234,68/158/174/234/282/297/346, 68/158/215/297/346, 68/215/297/346, 68/234,68/282/297/346, 68/297/346, 74/136/174/282/346, 74/136/174/297/346,74/136/346, 74/158/255/297, 74/255/346, 74/346,136/158/190/215/255/297/346, 136/158/215/297/346,136/174/215/255/282/297/346, 136/190/215/297/346, 136/215/234/282/297,136/215/234/297/346, 136/215/297, 136/297/346, 158/215/255/346,158/215/346, 170/203/254/296/343/346, 170/203/254/343/373,170/203/343/346, 174/190/234/297/346, 174/215/234/297/346,174/215/255/297/346, 174/282/297/346, 190/255/282/346, 190/297/346,203/296, 203/343, 203/343/346, 203/346, 215/255/297/346,215/234/297/346, 215/255/297/346, 215/297, 215/297/346, 215/346,234/255/346, 255/297/346, 255/346, 297/346, 343/346/373, and 346,wherein said positions are numbered with reference to SEQ ID NO:2. 4.The engineered polynucleotide of claim 1, wherein said engineeredpolynucleotide encodes an engineered transglutaminase comprising apolypeptide sequence having at least 90% sequence identity to SEQ IDNO:2, 6, 34, and/or
 256. 5. The engineered polynucleotide of claim 1,wherein said engineered polynucleotide encodes an engineeredtransglutaminase comprising a polypeptide sequence having at least 95%sequence identity to SEQ ID NO:2, 6, 34, and/or
 256. 6. The engineeredpolynucleotide of claim 1, wherein said engineered polynucleotideencodes an engineered transglutaminase comprising a polypeptide sequenceset forth in SEQ ID NO:2, 6, 34, or
 256. 7. The engineeredpolynucleotide of claim 1, wherein said engineered polynucleotideencodes an engineered transglutaminase comprising a polypeptide sequenceencoding a variant provided in Table 8.1, 9.1, 9.2, 10.1, and/or 11.1.8. The engineered polynucleotide of claim 1, wherein said engineeredpolynucleotide encodes an engineered transglutaminase comprising apolypeptide sequence selected from the even-numbered sequences set forthin SEQ ID NOS: 4 to
 756. 9. An engineered polynucleotide comprising apolynucleotide sequence that is at least 85% or more identical to asequence selected from the odd-numbered sequences set forth in SEQ IDNOS: 3 to
 755. 10. A vector comprising the engineered polynucleotidesequence of claim
 1. 11. A vector comprising the engineeredpolynucleotide sequence of claim
 9. 12. The vector of claim 10, furthercomprising at least one control sequence.
 13. The vector of claim 11,further comprising at least one control sequence.
 14. A host cellcomprising the vector of claim
 10. 15. A host cell comprising the vectorof claim 11.